Impact of Exercise-Induced Muscle Damage on Human Performance During Dynamic Exercise

IMPACT OF EXERCISE-INDUCED MUSCLE DAMAGE ON HUMAN

PERFORMANCE DURING DYNAMIC EXERCISE

CHAPTER ONE

INTRODUCTION

1.1 Introduction

Exercise-induced muscle damage (EIMD) is a commonly experienced phenomenon

resulting from unaccustomed exercise, particularly exercise with a high eccentric

component. Eccentric contractions involve the active lengthening of the muscle and occur

in every day activities such as walking downstairs. Many athletic activities also involve

eccentric muscle action, particularly during the landing or impact phase of running,

jumping or turning. Compared to concentric (shortening) and isometric (static)

contractions, eccentric muscle action is mechanically more efficient but employs a unique

activation strategy which predisposes the muscle to damage (Enoka, 1996; McHugh et al.,

2000).

1.2 Mechanisms of muscle damage

Proske and Morgan (2001) have postulated the initial series of events to explain how

muscle damage results from eccentric exercise (Figure 1.1). The model suggests that

during eccentric contractions weaker sarcomeres are stretched until they are beyond

myofilament overlap. This is understood to occur during the descending limb of the length-

tension curve where non-uniformity in sarcomere length is known to develop (see Figures

1.2 & 1.3) (Gordon et al., 1966). This leads to sarcomere disruption which is then followed

by membrane damage and subsequent dysfunction of the excitation-contraction (E-C)

mechanism. Muscle biopsy data provide evidence of disrupted sarcomeres, including Z-

line streaming (Newham et al., 1983a; Fridén, 1984), and of damage to t-tubules,

sarcoplasmic reticulum and sarcolemma (Lieber et al., 1994, 1996; Fridén & Lieber, 1998).

Disrupted

Sarcomeres

Overstretched

Sarcomeres

Membrane

Damage

Fibre DiesLocal

Contracture

Normal

Sarcomeres

Eccentric

Contractions

Shift in Optimum

Length E-C Coupling

Dysfunction

Rise in Passive

Tension

Delayed fall in

Tension

(Non-Reversible)

Fall in Tension

(Reversible) Swelling and

Soreness

Figure 1.1 Postulated series of events leading to muscle damage from eccentric exercise

(Proske & Morgan, 2001, p. 334).

This disruption is reported to occur predominantly in type II fibres, suggesting that they are

preferentially damaged (Fridén et al., 1983; Fridén & Lieber, 1992). More recently,

intravital microscopy observations have also revealed substantial microvascular

dysfunction including an increase in non-red blood cell-flowing capillaries and enlarged

capillary diameters (Kano et al., 2005). These ultrastructural disturbances initiate an influx

of Ca2+ into the sarcoplasm which activates proteolytic pathways involved in muscle fibre

break-down and repair (Peake et al., 2005, Tidball, 2005). These events in turn produce

symptoms associated with muscle damage, including increased intramuscular proteins in

the bloodstream (Hortobágyi & Denahan, 1989), prolonged loss of muscular strength,

decreased range of motion and increased muscle soreness (Clarkson et al., 1992; Cleak &

Eston, 1992).

Descending limb

Sarcomere length (μ)

Figure 1.2 The relationship between length and tension in skeletal muscle. Adapted

from Gordon et al. (1966), p.185.

Figure 1.3 Critical stages in the increase of myofilament overlap corresponding to key

points (1–6) labelled on the length-tension curve in figure 2.1. Adapted from Gordon et al.

(1966), p.186.

% maximal tension

1.3 Muscle function following eccentric exercise

Although there are several symptoms associated with EIMD, it has been suggested that

measurements of muscle function provide the best means of assessing the magnitude and

duration of muscle injury (Warren et al., 1999). Furthermore, the immediate and prolonged

loss of force-generating capacity that results from eccentric exercise is possibly the most

important symptom when considering the influence of EIMD on the human response to

dynamic exercise (Byrne et al., 2004).

Isometric strength measured at a fixed joint angle is the most frequently used assessment of

muscle function following eccentric exercise (Warren et al., 1999), with the greatest

decrements in maximal voluntary contraction (MVC) occurring immediately after eccentric

exercise, followed by a linear recovery (Byrne et al., 2001). Evaluation of dynamic muscle

function following EIMD can be achieved using isokinetic dynamometry. Although it is

not possible to replicate sport-specific movement patterns and velocities, insights into

dynamic muscle function have been gained using this technique. The magnitude of

strength loss and rate of recovery between isometric, concentric and eccentric muscle

actions appears to be similar following EIMD (Byrne & Eston, 2002a; Michaut et al.,

2002). However, conflicting findings have been reported in relation to changes in peak

torque at different angular velocities. Observations that decrements in peak torque are

greatest at higher angular velocities (Fridén et al., 1983; Eston et al., 1996) support the

notion that type II fibres may be selectively damaged during eccentric exercise (Fridén et

al., 1983; Fridén & Lieber, 1992); but are countered by conflicting evidence that peak

torque is affected to a greater extent at slower rather than faster angular velocities (Gibala

et al., 1995; Deschenes et al., 2000; Michaut et al., 2002).

1.4 Dynamic exercise

Athletic performance that relies on the muscle’s ability to generate force rapidly is

negatively impacted following exercise-induced muscle damage (EIMD). The loss of

force-generating capacity results in impaired sprint performances resulting from reductions

in peak power and increases in time to reach peak power. Immediate and prolonged

reductions have been observed in peak power output on a cycle ergometer (Sargeant &

Dolan, 1987; Byrne & Eston, 2002b), in time to peak power (Twist & Eston, 2007) and in

cycle sprint performance (Twist & Eston, 2005). However, recovery is not immediate and

further decrements at 24 and 48 h are observed, suggesting that delayed onset muscle

soreness (DOMS) may influence the dynamic response.

The influence of EIMD on endurance performance remains in dispute. Some studies have

reported no change in sub-maximal running performance following a prior bout of eccentric

muscle-damaging exercise (Hamill et al., 1991; Scott et al., 2003; Paschalis et al., 2005;

Marcora & Bosio, 2007). Whereas other investigations have observed that EIMD has a

negative effect on performance (Braun & Dutto, 2003; Chen et al., 2007b). EIMD does not

appear to alter the V˙

response to sub-maximal cycling (Gleeson et al., 1995, 1998;

Walsh et al., 2001; Moysi et al., 2005; Schneider et al., 2007); however the influence of

EIMD on other cycling performance measures is uncertain.

1.5 Summary

The intention of this thesis is to investigate the effects of exercise-induced muscle damage

(EIMD) on the response to dynamic exercise in human subjects. This topic has been

investigated infrequently and findings are equivocal.

Therefore, the main objective is to

provide empirical evidence to advance the scientific knowledge and understanding of the

phenomenon of EIMD; principally by investigating the physiological, perceived exertion

and metabolic responses to the performance of dynamic exercise with EIMD. This thesis

comprises 4 studies, as outlined below. Studies 1 and 4 used a total sample of 13

participants, 7 of which were common to both studies. Similarly, studies 2 and 3 used a

total sample of 16 participants, 7 of which were common to both studies.

Study 1 The effect of exercise-induced muscle damage on ventilatory and

perceived exertion responses to moderate and severe intensity cycle exercise

This study examined the effect of exercise-induced muscle damage (EIMD) on ventilatory

and perceived exertion responses to cycle exercise. Ten healthy, physically active men

cycled for six minutes at moderate intensity and to exhaustion at severe intensity before and

48 h after eccentric exercise (100 squats with a load corresponding to 70% of body mass).

Changes in ventilation and ratings of perceived exertion (RPE) were calculated for each

individual and expressed against time (moderate and severe exercise) and as a percentage

of time to exhaustion (severe exercise). Ventilation increased during moderate exercise at

48 h ( V˙

; 34.5 ± 5.0 to 36.3 ± 3.8 L.min-1, P<0.05) but increases in RPE were

not

significant.

During severe exercise at 48 h, time to exhaustion (TTE) was reduced and V˙ E

(87.1 ± 14.1 to 93.8 ± 11.7 L.min-1) and RPE (15.5 ± 1.3 to 16.1 ± 1.4) were elevated

(P<0.05). When expressed as a percentage of TTE, the differences in ventilation and RPE

values disappeared. Findings indicate that the augmented ventilatory response to cycle

exercise following EIMD may be an important cue in informing effort perception during

high intensity exercise but not during moderate intensity exercise.

No change was observed in the blood lactate response, although the ventilatory response

was enhanced, indicating that other ventilatory stimuli may play an important role

following EIMD. The potential dissociation of ventilatory and blood lactate responses

during ramp incremental cycling following EIMD was investigated in study 2.

This study has formed the basis of the following publication:

Davies, R. C., Rowlands, A. V., & Eston, R. G. (2009). Effect of exercise-induced muscle

damage on ventilatory and perceived exertion responses to moderate and severe intensity

cycle exercise. European Journal of Applied Physiology, 107(1), 11-19.

Study 2 The effect of eccentric exercise-induced muscle damage on the gas

exchange threshold

A prior bout of eccentric muscle-damaging exercise augments the ventilatory response to

constant-load cycle exercise without altering the blood lactate profile. However, the

influence of exercise-induced muscle damage (EIMD) on the performance of ramp

incremental exercise has received sparse attention. This study tested the hypothesis that

EIMD would augment the ventilatory response to subsequent ramp incremental cycle

exercise leading to a reduction in the gas exchange threshold (GET). In the absence of an

altered blood lactate profile this would indicate a dissociation of the GET from the lactate

threshold. Ten healthy, physically active male subjects (age, 25 ± 7 years; mass, 80.1 ±9.9

kg; height, 1.80 ± 0.08 m) performed maximal incremental cycle exercise tests before (pre)

and 48 h after (post) completing 100 squats, with a load corresponding to 70% body mass.

Following eccentric exercise GET occurred earlier (pre GET V˙ O2 : 1.58 ± 0.26; post

GET

V˙ O : 1.41 ± 0.14 l.min-1) while the blood lactate response was unchanged (P > 0.05). The

dissociation between GET and the blood lactate response during cycling with EIMD

indicates that the two phenomena are not causally linked. We propose that the 13%

increase in ventilation and resultant reduction in GET are evoked predominantly by

increased activation of group III and IV afferents which are stimulated via the mechanical

disruption of muscle fibres and local microvasculature due to eccentric exercise.

The decreased endurance capacity observed may have resulted from a shift in the muscle

metabolic profile to an increased reliance on non-oxidative metabolism. Therefore, the

purpose of study 3 was to investigate alterations in muscle metabolism during incremental

exercise to exhaustion.

This study is currently under review for publication:

Davies R.C., Rowlands A.V., Poole D.C., Jones A.M. and Eston R.G. Exercise-induced

muscle damage dissociates the Lactate and Gas exchange thresholds. Currently under

review.

Study 3 The 31P-MRS metabolic response to incremental exercise following

eccentric, muscle-damaging exercise

Performance decrements associated with EIMD include a reduction in maximal force-

generating capacity (Clarkson et al., 1992) and a shorter time to exhaustion (Asp et al.,

1998; Carmichael et al., 2005, 2006). Asp et al. (1998) have proposed that EIMD results in

a shift to more glycolytic energy production which may contribute to the accelerated

development of fatigue. This study investigated the influence of EIMD on changes in

muscle metabolism during incremental exercise using 31phosphorus magnetic resonance

spectroscopy (31P-MRS). Before and 48 h after performing100 squats, 31P-MRS was used

to measure dynamic changes in [PCr], [Pi], [ADP] and pH during knee-extensor

incremental tests to exhaustion. The resting [Pi]:[PCr] ratio was increased 48 h after

eccentric exercise (pre: 0.12 ± 0.02; post: 0.18 ± 0.05). During incremental exercise the

changes in pH, [PCr] and [Pi]:[PCr] were similar but did not continue for as long. Time to

exhaustion and associated peak work rate values were significantly reduced following

eccentric exercise (pre: 519 ± 56; post: 459 ± 63 s) and (pre: 29 ± 4; post: 25 ± 4 W),

respectively. End exercise pH and [PCr] values were significantly higher (pre: 6.75 ± 0.12;

post: 6.83 ± 0.15) and (pre: 20 ± 13; post: 33 ± 15 % baseline values), respectively. These

findings demonstrated that the accelerated development of fatigue following eccentric

exercise did not result from alterations in muscle phosphate metabolism.

While phosphate metabolism does not appear to have a detrimental influence on the

performance of dynamic exercise, the substantial microvascular dysfunction observed

following EIMD (Kano et al., 2005) may contribute to impaired performance due to

disruptions to delivery and distribution of O2 within the capillary bed of the active muscle.

Thus study 4 was designed to investigate the influence of EIMD on the matching of O2

delivery to O2 utilisation.

This study is currently under review for publication:

Davies RC, Eston RG, Fulford J, Rowlands AV and Jones AM. Muscle damage alters the

metabolic response to dynamic exercise in humans: a 31P-MRS study. Currently under

review.

Study 4 The effect of eccentric exercise-induced muscle damage on the

dynamics of muscle oxygenation and pulmonary oxygen uptake

Unaccustomed eccentric exercise has a profound impact on muscle structure and function.

However, it is not known whether associated microvascular dysfunction disrupts the

matching of

O2 delivery ( Q˙ O2 ) to O2 utilisation ( V˙ O2 ). Near infra-red spectroscopy

(NIRS) was used to test the hypothesis that eccentric exercise-induced muscle damage

would

elevate

the

muscle Q˙ O2 : V˙ O2 ratio

during

severe

intensity exercise

whilst

preserving the speed of the V˙ O2 kinetics at exercise onset. Nine physically active men

completed ‘step’ tests to severe-intensity exercise from an unloaded baseline on a cycle

ergometer before and 48 h after eccentric exercise (100 squats with a load corresponding to

70%

body mass).

NIRS

and

breath-by-breath

pulmonary V˙ O2 were measured

continuously during the exercise tests and subsequently modelled using standard non-linear

regression

techniques. There

were

changes

phase

II pulmonary V˙ O2 kinetics

following the onset of exercise (time constant, pre: 25 ± 4; post: 24 ± 2 s; amplitude, pre:

2.36 ± 0.23; post: 2.37 ± 0.23 L/min; all P>0.05). However, the primary (pre: 14 ± 3; post:

19 ± 3 s) and overall (pre: 16 ± 4; post: 21 ± 4 s) mean response time of the [HHb] response

was significantly slower following eccentric exercise (P<0.05). The slower [HHb] kinetics

observed following eccentric exercise is consistent with an increased

Q˙ O2 : V˙ O2 ratio

during transitions to severe-intensity exercise. We propose that unchanged primary phase

V˙ O2 kinetics are associated with an elevated Q˙ O2 : V˙ O2 ratio that preserves blood-

myocyte O2 flux.

This study has formed the basis of the following publications/presentations:

Publication:

Davies, R. C., Eston, R. G., Poole, D. C., Rowlands, A. V., Dimenna, F., Wilkerson, D. P.,

et al. (2008). The effect of eccentric exercise-induced muscle damage on the dynamics of

muscle oxygenation and pulmonary oxygen uptake. Journal of Applied Physiology, 105(5),

1387-1388.

Because the reviewers thought the paper could have substantial impact on the field, Dr.

Mark Burnley was invited to write a Commentary to accompany the publication of the

article:

Burnley M. (2008). Found in translation: the dependence of oxygen uptake kinetics on O 2

delivery and O2 utilization. Journal of Applied Physiology, 105(5), 1387-1388

Presentation

Davies R, Eston R, Poole D, Rowlands A, Dimenna F, Wilkerson D, Twist C, and Jones A.

(2008). The influence of exercise-induced muscle damage on pulmonary oxygen uptake

and muscle deoxygenation kinetics during high intensity exercise. In J. Cabri, F. Alves, D.

Araujo, J. Barreiros and A. Veloso (Eds), 13th Annual Congress of the European Congress

of Sports Sciences, Book of Abstracts (p630). Estoril.

CHAPTER TWO

REVIEW OF THE LITERATURE

2.1 Introduction

The temporary damage to skeletal muscle that results from unaccustomed exercise has been

the subject of investigation by exercise physiologists for over 100 years. In 1902 Hough

described the delayed but transient soreness experienced when untrained muscle made a

series of contractions against a strong spring, suggesting that it resulted from ruptures

within the muscle. Since then, several hundred published investigations have attempted to

elucidate the underlying mechanisms of this phenomenon and document its symptoms.

Direct histological analysis of both human and animal muscle tissue has provided evidence

to support Hough’s (1902) original contention that changes in skeletal muscle morphology

are symptomatic of unaccustomed exercise.

2.2 Changes in skeletal muscle morphology

Human muscle biopsy data has provided direct evidence of considerable disruption in the

ultrastructure of skeletal muscle following unaccustomed eccentric exercise (Fridén et al.,

1981, 1983; Fridén, 1984; Newham et al., 1983a; Jones et al., 1986; Gibala et al., 1995).

Disturbances which originate in the myofibrilar Z-band are seen as streaming, broadening

or total disruption. Z-line disruption is the most frequently reported ultrastructural

abnormality and as such may represent the weak link in the myofibril contractile

mechanism (Newham et al., 1983a; Fridén 1984). Reports of greater disruption of type II

fibres have led to the understanding that these fibres are preferentially damaged during

eccentric exercise (Fridén et al., 1983; Fridén , 1984; Jones et al., 1986 Lieber et al., 1991;

MacPherson et al., 1996). Eccentric contractions are understood to generate higher levels

of mechanical stress than concentric or isometric contraction due to reduced motor unit

activation (Enoka, 1996; Armstrong et al., 1991; McHugh et al., 2000).

This has led to

speculation that type II motor units are selectively recruited during eccentric contraction

(Enoka, 1996; McHugh et al., 2000, 2002; Nardone & Schieppati, 1988; Nardone et al.,

1989; Howell et al., 1995) and that the excessive stress on the small number of active fibres

leads to them becoming damaged (McHugh et al., 2000).

Figure 2.1

Longitudinal sections of fast-twitch (FT) fibres in A) triceps brachii muscle

of a sedentary control rat, and B) rat triceps brachii muscle 1 day after exercise downhill

running (DH). Scale bars, 1μm. Adapted from Takekura et al. (2001). Note the Z-line

smearing and focal disruption of the A-band region following downhill running.

In animal models used to study EIMD, damage to contractile and cytoskeletal components

of predominantly type II fibres has been revealed using histological staining (Fridén and

Lieber, 1992; Takekura et al., 2001) (Figure 2.1). Lack of staining for cytoskeletal proteins

has provided evidence of disruption to the sarcolemma and cytoskeleton (Lieber et al.,

1994, 1996; Fridén & Lieber, 1998; Komulainen et al., 1998, 1999, 2000). Morphological

changes in skeletal muscle also include ultrastructural changes in the arrangement of the T-

tubules which is believed to occur when adjacent myofilaments show disparate degrees of

stretch (Takekura et al., 2001; Yueng et al., 2002), and the presence of multiple central

nuclei (Kano et al., 2004). Furthermore, disruption of capillary geometry including

enlarged capillary diameters has been observed using intravital microscopy, possibly

resultant to myocyte swelling (Kano et al., 2004).

In human studies changes in the intermediate filament system have been interpreted as

evidence of myofibrillar and cytoskeletal damage (Fridén et al., 1984). However, this

interpretation has been challenged by Yu and colleagues (2002) who have reported

increased rather than decreased staining of cytoskeletal proteins. These authors suggested

that their findings provide evidence of muscle repair and remodelling rather than muscle

damage from eccentric contraction (Yu et al., 2002, Yu & Thornell, 2002). Whilst animal

models may not always accurately reflect changes in their human counterparts, they have

provided valuable insights which are not so easily procured in human models. Indeed,

caution should be taken when interpreting the results of studies using human muscle biopsy

as there is evidence to suggest that the biopsy procedure itself may produce some changes

mistakenly attributed to EIMD (Malm et al., 2000; Roth et al., 2000). Nonetheless direct

analysis of muscle tissue has revealed substantial disruption in both human and animal

models of EIMD.

2.3 Indirect markers of muscle damage

Due to the invasive nature of muscle biopsy procedures in human investigations research

scientists have increasingly selected to utilise indirect markers of muscle damage in an

attempt to further our understanding of the underlying mechanisms of EIMD and its

symptoms.

2.3.1 Muscle protein efflux

The structural and functional status of muscle tissue can be determined by the level of

skeletal muscle enzymes in the blood. If sarcolemmal integrity is compromised as a result

of eccentric exercise (as reported in 2.2), an efflux of muscle proteins into the bloodstream

will result (Hortobágyi & Denahan, 1989). Muscle proteins such as creatine kinase (CK)

and lactate dehydrogenase (LDH) are commonly used as indirect markers of muscle

damage. In fact, Warren et al. (1999) reported that, of human studies reviewed, over 50%

used changes in blood levels of myofibre proteins as evidence of EIMD with CK being the

most frequently reported. However, the time course of CK activity in the blood appears to

be dependant on the damage protocol employed. Following muscle-damaging exercise

such as downhill running, weightlifting or plyometric exercise, plasma CK levels peak at

24-48 h (Paul et al., 1989; Eston et al., 1996; Horita et al., 1999; Byrne and Eston, 2002a;

Twist & Eston, 2005; Chen et al., 2007b). In contrast, high-force eccentric exercise using

isokinetic dynamometers induces a delayed response with CK levels peaking at day 4-5

(Clarkson et al., 1992; Nosaka & Clarkson, 1992, Chen et al., 2003, Zainuddin et al., 2005).

Interpretation of the CK response to eccentric exercise is further complicated by high inter-

subject variability despite similar decrements in contractile function (Clarkson and

Ebbeling, 1988; Hortobágyi and Denahan, 1989). Whilst it is compelling to hypothesise a

relationship between the loss of sarcolemmal integrity, increase in plasma CK activity and

loss of muscle function, there is no evidence that plasma CK levels accurately reflect the

extent of myofibre damage caused (Nosaka & Clarkson, 1992).

2.3.2 Calcium homeostasis

Myofibril damage in animal models of EIMD has been associated with the loss in calcium

(Ca2+) homeostasis due to disturbances of the myocyte membrane (Armstrong, 1990). The

disruption of sarcoplasmic reticulum (SR) (Byrd, 1992; Fridén & Lieber, 1996) increases

membrane permeability and is understood to be responsible for increases in intracellular

Ca2+ concentration (Armstrong, 1984). Increases in Ca2+ may then contribute to the further

degradation of the muscle tissue by stimulating the release of calcium-activated neutral

proteases such as calpain which have been shown to damage Z-line-associated proteins

(Busch et al., 1972; Belcastro, 1993; Belcastro et al., 1998). However, the direct

investigation of SR regulation of calcium in human EIMD is extremely limited and has

produced conflicting results. Nielson et al. (2005) observed no change in SR function

following eccentric, muscle-damaging exercise. In contrast, Enns and colleagues (1999)

reported no immediate change in SR Ca2+ uptake but a prolonged alteration in SR function

in the 2-14 day recovery period. The administration of calcium channel blockers (CCB) in

animal models of EIMD has been reported to reduce or prevent the rise in intracellular Ca2+

and subsequent injury (Soza et al., 1986; Duan et al., 1990; Duarte et al., 1992; Armstrong

et al., 1993). Similarly, in humans, damage to some sarcomeric proteins was attenuated or

delayed by the administration of CCBs following eccentric exercise (Beaton et al., 2002).

However, the administration of CCBs unexpectedly increased the infiltration of

inflammatory cells including neutrophils and macrophages into the muscle tissue, possibly

due to the influence of CCBs on vascular and smooth muscle tone (Beaton et al., 2002).

2.3.3 Inflammatory response

Following eccentric, muscle-damaging exercise, inflammatory cells such as neutrophils and

macrophages are understood to infiltrate the muscle in order to remove necrotic tissue and

initiate the process of muscle repair and regeneration (MacIntyre et al., 1995; Peake et al.,

2005; Tidball, 2005). The infiltration of these inflammatory cells has also been implicated

in producing secondary cytoskeletal disruptions to eccentrically exercised muscle (Pizza et

al., 2001, 2005)

The first inflammatory cells to accumulate are neutrophils (Fielding et al., 1993; Malm et

al., 2000). These cells remove necrotic tissue by phagocytosis and release cytokines to

attract additional inflammatory cells. Neutrophils can permeate human skeletal muscle

within an hour of eccentric exercise and remain present for up to 5 days (Fielding et al.,

1993) although significant increases are more commonly reported to last up to 24 h

(MacIntyre et al., 1996, 2000, 2001, Beaton et al., 2002). Neutrophil accumulation is

understood to activate resident macrophages and attract further macrophage invasion.

Macrophages are not only actively phagocytic but may also promote repair and

regeneration via the release of cytokines known to cause myoblast proliferation in vitro

(Hawke & Garry, 2001). However, a recent review has revealed that only 55% of human

studies, as opposed to 85% of animal studies, have detected neutrophil infiltration in

exercise-damaged muscle (Schneider & Tiidus, 2007). Animal models of EIMD are most

commonly used to study inflammatory cell accumulation due to the difficulties inherent in

multiple biopsy procedure in human subjects (Pizza et al., 2008). Thus the inflammatory

response to eccentric exercise in humans remains controversial, not least because it appears

to be dependent on a wide variety of factors including the mode, intensity and duration of

exercise, the muscle groups examined and the method of detection (directly using muscle

biopsy or indirectly via blood analysis) (Peake et al., 2005; Tidball, 2005; Schneider &

Tiidus, 2007).

2.3.4 Impaired metabolism

A number of studies have indicated that intramuscular glycogen stores are depleted

following eccentric, muscle damaging exercise. O’Reilly et al. (1987) demonstrated a

prolonged depletion of muscle glycogen content following 45 min of eccentric cycling.

Muscle biopsy samples showed that muscle glycogen content had dropped to 61% of

baseline values immediately after eccentric exercise and were further depleted to 44% of

baseline values 10 days after the exercise bout. Subsequent studies have corroborated these

findings which indicate that EIMD may impair muscle glycogen resynthesis (Asp et al.,

1995; Asp et al., 1998; Costill et al., 1990).

Resting muscle glycogen uptake is decreased following eccentric exercise due to a decrease

in insulin sensitivity. The transient insulin resistance reported following eccentric exercise

(Kirwan et al., 1992; del Aguila et al., 2000; Asp et al., 1996) has been linked to the

decrease in a major glucose transport protein, GLUT-4 (Asp et al., 1995; Asp et al., 1996).

The translocation of GLUT-4 to the cell membrane when additional glucose is required is

initiated by the binding of insulin to membrane-bound insulin receptors (IRS-1) (Tee et al.,

2007). The physiological stress associated with EIMD appears to impair the insulin

stimulation of IRS-1 and the subsequent activation of GLUT-4, leading to decreased

insulin-mediated glucose uptake (del Aguila et al., 2000).

While type I muscle fibres are predominantly oxidative, type II fibres, which are selectively

recruited (Enoka, 1996; McHugh et al., 2000, 2002; Nardone & Schieppati, 1988; Nardone

et al., 1989; Howell et al., 1995) and preferentially damaged (Fridén et al., 1983; Fridén ,

1984; Jones et al., 1986 Lieber et al., 1991; MacPherson et al., 1996) during eccentric

contractions, are predominantly glycolytic. Asp et al. (1998) have reported that the resting

glycogen content of type II fibres is more severely depleted than that of type I fibres

following eccentric exercise (Asp et al., 1998). This observation has led to speculation that

increased glygogenolysis may result from EIMD and could also be responsible for the

higher resting blood lactate concentration [La] reported with EIMD (Asp et al., 1996, Asp

et al., 1998). Elevated [La] has also been reported during exercise with EIMD and has

similarly been attributed to a shift to more glycolytic energy production (Braun & Dutto,

2003; Chen at al., 2007b, 2008; Gleeson et al., 1995, 1998). It has been suggested that

damage to type II fibres would necessitate a greater recruitment of these fibres during

exercise with EIMD in order to maintain the required force production (Gleeson et al.,

1998). However, higher [La] does not appear to be an obligatory consequence of EIMD.

Several studies have reported no change in the [La] response to exercise following prior

eccentric exercise (Hamill et al., 1991; Scott et al., 2003; Marcora & Bosio, 2007; Moysi et

al., 2005). A recent study by Schneider et al. (2007) reported unchanged phase II V˙ O2

kinetics indicating that EIMD did not compromise oxidative function. These authors

suggested that the increase in [La] observed must therefore result from an increased lactate

efflux from the active muscle due to increased membrane permeability following muscle-

damaging exercise (Schneider et al., 2007).

2.3.5 Delayed onset muscle soreness

Delayed onset muscle soreness (DOMS) was first described by Hough (1902) and is the

characteristic manifestation most commonly associated with EIMD. In a review of

measurement tools used to assess EIMD, Warren et al. (1999) reported that subjective and

objective assessment of DOMS was reported in 63% and 12% of the human studies

reviewed, respectively; the most frequently observed measurement of muscle damage

employed (Warren et al., 1999). However, a poor temporal relationship exists between

DOMS and changes in muscle morphology (Newham et al., 1983a; Jones et al., 1986) and

changes in muscle function (Newham et al., 1983b; Howell et al., 1993; Rodenburg et al.,

1993; Saxton et al., 1995; Prasartwuth et al., 2005). The ‘delay’ in the experience of

soreness appears to vary amongst individuals but peak soreness generally develops between

24 and 48 h after the muscle-damaging exercise (Newham et al., 1983b, 1988; Jones et al.,

1987, 1989; Clarkson et al., 1992; Cleak & Eston, 1992) and gradually subsides, usually

disappearing within 96 h (Jones et al., 1987; Cleak & Eston 1992). In contrast, changes in

muscle morphology were found immediately after completion of a 20 min step-test with the

severity of the damage increasing in the biopsy samples taken at 24 and 48 hours (Newham

et al., 1983a). In an earlier investigation Fridén and colleagues (1981) demonstrated that

ultrastructural disturbances were 3 times greater in biopsy samples taken 2 as opposed to 7

days after the eccentric exercise bout although no biopsy was taken immediately after the

damage protocol which involved repeatedly running downstairs (Fridén et al., 1981).

Functional impairments, in particular changes in strength also follow a different time

course to the development of DOMS (Rodenburg et al., 1993; Newham et al., 1983b:

Nosaka et al., 2002). Strength decrements are greatest immediately after eccentric exercise

and demonstrate a linear recovery back to baseline measurements (Clarkson et al., 1992;

Sayers & Clarkson, 2001; Howell et al., 1993; Byrne & Eston, 2002a) usually within 5-7

days (Armstrong, 1984; Clarkson & Tremblay 1988) although in some individuals,

recovery may take up to several weeks (Clarkson et al., 1992; Howell et al., 1993). Thus

DOMS is a poor indicator of both functional impairment and the magnitude of

morphological muscle damage.

Several theories have been proposed to explain the mechanisms responsible for the

sensation of DOMS, including the muscle damage theory originally presented by Hough

over 100 years ago (Hough, 1902). The mechanical disruption of structural elements, (for

further detail see section 2.2) is believed to contribute to the stimulation of pain receptors

(nociceptors). These are primarily group III and IV thin-fibre afferent neurons, located in

the muscle, connective tissue, musculotendinous junction, arterioles and capillaries and

their stimulation is understood to lead to the sensation of pain (Cheung et al., 2003).

Several studies have demonstrated a relationship between increases in muscle soreness and

impairments in dynamic muscle function (Horita et al., 1999; Proske et al., 2003;

Weerokkody et al., 2003b). However, as explained above, differences in the temporal

relationship between DOMS and mechanical disruption indicate that the muscle damage

theory can only partially explain the development of DOMS. The noxious stimulus of

lactic acid has been implicated in producing the sensation of DOMS (Armstrong, 1984).

Elevations in blood lactate concentration [La] have been reported in several studies during

exercise following EIMD (Gleeson et al., 1995, 1998; Braun & Dutto, 2003). However, the

lactic acid theory has largely been rejected as there appears to be no relationship between

ratings of soreness and blood [La] levels following a bout of downhill running designed to

induce EIMD (Schwane et al., 1983) and higher levels of lactate have been shown not to

induce soreness in concentric exercise (Armstrong, 1984; Schwane et al., 1983).

Inflammation and swelling triggered by the damage is presently the most widely accepted

proposed mechanism for DOMS (Smith, 1991, Proske & Morgan, 2001; Cheung et al.,

2003). The pain or soreness resulting from eccentric exercise manifests itself as a dull

aching pain that is stimulated by either palpation or contraction and is not present at rest

(Cleak & Eston, 1992; Avela et al., 1999; Komi, 2000). Several studies have proposed that

swelling subsequent to EIMD may be mechanistically implicated in the development of

DOMS due to increases in local tissue pressure (Howell et al., 1985; Bobbert et al., 1986;

Fridén et al., 1986). Smith (1991) has hypothesised that increases in intramuscular pressure

during contraction or palpation would provide sufficient mechanical stimulus for the

sensitisation of mechanical nociceptors (Smith, 1991). These thin-fibre afferents are also

known to be sensitised by various inflammatory mediators including bradykinins,

prostaglandins and histamines which are released during the process of proteolytic

breakdown and repair (Clarkson & Hubal, 2002). It has been suggested that the delay in

the inflammatory response may account for the delay in the symptoms of DOMS (Smith,

1991). However, Taguchi and colleagues (2005) have demonstrated that chemical stimuli

including pH 5.5, adenosine triphosphate, and bradykinin, do not influence the response of

group III and IV muscle afferents to eccentric exercise (Taguchi et al., 2005).

2.3.6 Changes in strength

The immediate and prolonged loss of strength that occurs after eccentric exercise is one of

the most frequently used markers of EIMD. In a review of measurement tools used in

studies of EIMD, Warren et al. (1999) reported that 50% of human studies measured

maximal voluntary contraction (MVC) torque, a measure which the authors suggest

provides the most accurate and reliable indirect marker of muscle damage in human studies

(Warren et al., 1999). An immediate loss of force generating capacity may also be

observed following non-damaging concentric exercise but recovery to baseline strength

occurs within hours (Newham et al., 1983b; Jones et al., 1989). The greatest strength loss

and longest recovery times have been associated with high-force eccentric protocols such as

that employed by Newham and colleagues (1987) which involved maximal eccentric

contraction of the elbow flexors. These authors and others have reported strength

decrements of over 50% compared to pre-exercise values, with a linear recovery to baseline

typically lasting 1 - 2 weeks (Newham et al., 1987; Saxton et al., 1995; Nosaka et al., 1991)

although in some individuals, recovery may take up to several weeks (Clarkson et al., 1992;

Howell et al., 1993). In contrast, downhill running (Eston et al., 1996, Eston et al., 2000)

and bar-bell squatting protocols (Byrne & Eston 2002a, 2002b; Moysi et al., 2005)

designed to provoke muscle damage, typically generate a more modest loss in force-

generating capacity with strength decrements of between 10 and 30% and recovery to

baseline occurring within 4-7 days.

2.4 Muscle function following exercise-induced muscle damage

Morgan and Allen (1999) proposed that the earliest events in eccentric, muscle-damaging

exercise involve the over-stretching of randomly distributed sarcomeres, although the initial

decline in strength may be attributed to metabolic fatigue, damaged muscle or a

combination of both (Morgan & Allen 1999). If sarcomeres are stretched to a point of

minimal overlap between actin and myosin filaments, cross-bridge formation would be

reduced and the ability to generate force would be compromised (Clarkson et al., 1992). In

support of this hypothesis, it has been reported that loss in strength is greater when the

damaging exercise is performed with muscle at longer rather than shorter lengths (Newham

et al., 1988; Child et al., 1998).

2.4.1 Changes in optimal muscle length

A shift in the length-tension relationship for a given force to longer muscle length

following eccentric exercise was first observed by Katz (1939), who proposed that rapid

over-stretching of muscle beyond its optimum length was likely to damage the contractile

elements. Katz’s original (1939) findings based on frog and tortoise muscle have been

confirmed by more recent investigations using human models (Jones et al., 1997;

Whitehead et al., 1998; Brockett et al., 2001; Philippou et al., 2003) (Figure 2.2).

These findings provide evidence to support the thesis that a longer muscle length is needed

to achieve the same myofilament overlap after eccentric exercise due to an increase in

series compliance resulting from the over-stretching of sarcomeres (Morgan & Allen, 1999;

Proske & Morgan, 2001). In further support of this theory, a greater loss in strength is

reported at short as opposed to longer or optimal muscle length following eccentric exercise

indicating a shift towards longer muscle lengths for maximal force generation (Saxton &

Donnelly, 1996; Child et al., 1998; Byrne et al., 2001; Sayers & Clarkson, 2001; Byrne &

Eston, 2002b).

Figure 2.2 Hamstrings angle-torque curves before eccentric exercise (Control) (O) and

immediately post exercise (●). Gaussian curves have been fitted to the top 10% of each

curve. Adapted from Brockett et al., (2001). Note the immediate right shift of the angle-

torque relationship following eccentric exercise.

2.4.2 Low-frequency fatigue

An alternative hypothesis to explain the loss in force-generating capacity and the shift in

the length-tension relationship proposes that alterations in calcium homeostasis may be

responsible. A decrease in the release of calcium ions (Ca2+) from the sarcoplasmic

reticulum (Warren et al., 1993; Westerblad et al., 1993) and an increase in intracellular Ca2+

(Chin & Allen, 1996) has been shown to lead to failure of the excitation-contraction (E-C)

coupling process in mouse muscle preparations. In human biopsy samples Hill et al. (2001)

have reported that sarcoplasmic reticulum Ca2+ release and uptake were significantly

depressed following exercise that provoked decrements in knee extensor MVC. These and

other authors have also observed a significant correlation between decreases in torque

production at low electrical stimulation frequencies and Ca2+ release following exercise

(Hill et al., 2001, Neilsen et al., 2005).

The disproportionate loss of force at low (20Hz) compared with high (100Hz) frequencies

of electrical stimulation is known as low-frequency fatigue (LFF) and is likely caused by an

impairment of excitation-contraction coupling process (Edwards et al,. 1977). It is known

to be induced by fatiguing exercise (Edwards et al., 1977; Newham et al., 1983b; Jones et

al., 1989), with the most profound effect provoked by eccentric exercise (Newham et al.,

1983b; Jones et al., 1989). Thus there is evidence from both animal and human studies to

suggest that reductions in sarcoplasmic reticulum Ca2+ release are the primary cause for

LFF. However, there is evidence that, in addition to reduced Ca2+ release, LFF that follows

eccentric exercise may be caused by changes in muscle morphology and subsequent

remodelling (Jones et al., 1996; Westerblad et al., 2000). The redistribution of sarcomere

lengths following eccentric exercise may account for the length-dependent effect of

strength loss associated with EIMD, particularly in view of the fact that, like decrements in

MVC, LFF is more evident at short rather than long muscle lengths. (Jones et al., 1989;

Byrne et al., 2001)

2.4.3 Alterations to neural control

The generation of muscular force is not only a product of muscle contractile function and

E-C coupling but also of neural drive. Therefore the inability to generate maximal force in

muscles damaged by eccentric exercise could theoretically be the result of alterations in

neural

drive,

estimates

which

can

obtained

using

electromyography

(EMG).

Deschenes et al. (2000) have provided evidence to suggest that neuromuscular efficiency,

the ratio of torque generated to integrated EMG (iEMG) activity, is decreased following

EIMD. These authors reported increases in iEMG activity and a decrease in the torque:

iEMG ratio during maximal isometric contractions which persisted for 10 days. Other

EIMD symptoms including plasma CK activity, perceived soreness and peak torque were

all recovered within 7 days (Deschenes et al., 2000). Subsequent investigations have

reported similar disturbances in the torque:iEMG ratio with the largest effect apparent at

low forces; arguably where the precision of force production is most functionally relevant

(Weerakkody et al., 2003a; Lavender & Nosaka, 2006; Semmler et al., 2007). Increased

isometric force fluctuations have also been observed following eccentric, muscle-damaging

exercise but they appear not to be an artefact of muscle damage as measures return to

baseline levels within 24 h (Lavender & Nosaka, 2006; Semmler et al., 2007).

Alterations have also been reported in the perception of force production and joint position

following eccentric exercise. Saxton et al. (1995) demonstrated that following eccentric

exercise of the forearm flexors, participants consistently overestimated the amount of force

they could produce. Rather than matching the target force of 35% MVC generated in the

undamaged (control) arm, participants undershot their target force for the 5 days of the

study following the initial insult (Saxton et al., 1995). However, when the forces applied

by both damaged and control arm were expressed as a proportion of the MVC for that arm

at that time, errors in estimation were reduced or absent (Saxton et al., 1995). In addition to

disturbances in the matching of a sense of force, disturbances in the reproduction of limb or

joint position have been reported following eccentric exercise (Saxton et al., 1995; Brockett

et al., 1997; Walsh et al., 2004). However, conflicting results were recorded. Brockett et

al. (1997) reported that participants produced larger joint angles following eccentric

exercise whereas Saxton et al. (1995) and Walsh et al. (2004) reported that participants

produced smaller joint angles following eccentric exercise. It has been proposed that

alterations to the perception of force and limb position following eccentric exercise may be

due to damaged sensory receptors within the muscle (Saxton et al., 1995; Brockett et al.,

1997; Carson et al., 2002; Proske et al., 2003). It is believed the sense of force is derived

from peripheral receptors in the muscle and the Golgi tendon organs and that muscle

spindles provide the signals for position sense (McCloskey, 1978; Gandevia, 1996). Twist

et al. (2008) reported impaired unilateral balance performance 24 h after plyometric,

muscle damaging exercise which the authors attributed to alterations in proprioceptive

control. However recent work by Gregory and colleagues using anaesthetised cat models

has demonstrated that the responsiveness of the tendon organs and muscle spindles is not

altered by eccentric exercise (Gregory et al., 2002, 2004). Thus the effect of eccentric,

muscle damaging exercise on proprioceptive function is yet to be explained.

It has been proposed that the increases in iEMG associated with altered proprioception

following EIMD may be indicative of altered motor unit activation (Proske et al., 2004;

Weerokkody et al., 2003b; Prasartwuth et al., 2005; Semmler et al., 2007). Proske et al.

(2003) have suggested that DOMS may be involved in alterations to neural control

following eccentric exercise. They propose that the pain from DOMS leads to a reduced

motor cortical excitability, which may serve to protect muscle during from further damage

during the repair process (Proske et al., 2003). However, the mechanism or mechanisms by

which EIMD influences alterations in motor unit recruitment remains to be determined.

2.5 The Repeated Bout Effect

The protective adaptation to a single bout of eccentric exercise has been termed the

‘repeated bout effect’ (Nosaka & Clarkson, 1995). Whenever unaccustomed eccentric

exercise is repeated, within a given time-frame, the magnitude of the characteristic

symptoms of EIMD is diminished. Changes in muscle morphology, muscle protein efflux,

inflammation, loss of strength and other symptoms are attenuated by a repetition of the

same exercise but the damaging effects are not prevented (Nosaka & Clarkson, 1995;

McHugh et al., 1999; McHugh, 2003). The repeated bout effect may be conferred as early

as 2 days after the initial exercise bout (Paddon-Jones et al., 2000; Nosaka & Newton,

2002) and for most of the symptoms of EIMD, lasts at least 6 months but is lost between 9

and 12 months (Nosaka et al., 2001a). Very little prior exercise is needed to confer the

effect. As few as 2 maximal eccentric contractions have been demonstrated to confer

protection against symptoms of EIMD when the same elbow flexor muscles performed 24

maximal contractions 2 weeks later (Nosaka et al., 2001b). Similarly Lavender and Nosaka

(2008) showed that light eccentric exercise, 30 (6 x 5) contractions of just 10% MVC was

effective in attenuating muscle damage against a subsequent bout of eccentric exercise of

40% MVC performed 48 h later.

The mechanisms underlying the repeated bout effect are not fully understood although

several potential mechanisms have been proposed. It has been suggested that protection is

conferred as a result of neural, cellular and mechanical adaptations which may work

independently of each other or in concert (McHugh et al., 1999; McHugh 2003). The

neural adaptation theory proposes that during a subsequent bout of eccentric exercise,

motor unit recruitment is altered in order to redistribute the workload and thus improve

motor unit efficiency (Nosaka & Clarkson, 1995). Warren et al. (2000) observed a 30%

decrease in EMG mean frequency in tibialis anterior muscle in the second of two bouts of

50 eccentric MVCs separated by one week. These authors concluded that the data

indicated that an increased activation of slow motor units and a concomitant decrease in

activation of fast units occurred in the repeated bout (Warren et al., 2000). The findings of

Chen (2003) lend further support to the thesis of reduced activation of fast-twitch motor

units during the second eccentric bout. However, McHugh et al. (2001) were unable to find

any evidence of neural adaptation after performing a repeated bout of relatively low-

intensity eccentric exercise.

The cellular adaptation theory involves potential adaptations of the contractile machinery.

Proske and Morgan (2001) have suggested that following eccentric exercise optimum

muscle length increases due to the addition of sarcomeres in series. A shift of optimum

angle towards a longer muscle length following EIMD has been confirmed in several

studies (see section 2.4.3). However, in a recent study, Chen and colleagues (2007a)

revealed that while a rightward shift in optimum angle demonstrated a relationship with the

degree of muscle damage associated with the initial bout, it did not appear to be directly

related to the mechanisms responsible for the repeated bout effect (Chen et al., 2007a).

Several investigations have used blood markers to show that there is a reduction in the

inflammatory response associated with EIMD following a repeated bout of eccentric

exercise (Pizza et al., 1996, 2001; Hirose et al., 2004; Smith et al., 2007). However, a more

recent study using muscle biopsy, demonstrated that several inflammatory genes were

transcriptionally up-regulated (rather than attenuated) following a repeated bout of

eccentric leg exercise (Hubal et al., 2008).

Mechanical adaptation to eccentric exercise may involve the remodelling of the

intermediate filament system to provide mechanical reinforcement against subsequent

bouts. Yu and Thornell (2002) showed that a single bout of downstairs running increased

staining of actin and desmin, suggesting that this reflected an increased synthesis of the

proteins as part of an adaptation process. More recently Lehti and colleagues (2007) have

used rat biopsy data to demonstrate that prior eccentric exercise produced an adaptive

response that protected the sarcolemma, intermediate filament, and sarcomeric proteins

against subsequent disruption. While there may be several mechanisms underlying the

repeated bout effect which may work in isolation or to compliment each other, a unified

theory to explain the mechanism of protective adaptation remains elusive.

The recently revealed presence of cross-over or contralateral adaptation to eccentric

exercise has indicated that the repeated bout effect is likely to involve a complex interplay

of all three proposed mechanisms (Howatson & van Someren, 2007). Following the second

bout of 45 eccentric MVCs of the elbow flexors, symptoms of EIMD were diminished

when the second bout was performed with the opposite or contralateral limb although the

magnitude of change was not as profound as when the same arm was exercised twice. The

authors have suggested that the contralateral adaptation was most likely mediated by neural

mechanisms as there was no direct stimulus for cellular of mechanical changes to the

contralateral arm (Howatson & van Someren, 2007). Thus the more profound adaptation to

the same-side arm most likely results from neural, cellular and mechanical mechanisms

working in concert.

2.6 Dynamic muscle function

Of all the symptoms of EIMD, the immediate and prolonged impairment to muscle function

has the potential to be the most debilitating when considering the human response to

dynamic exercise following eccentric exercise. However, the study of dynamic muscle

function during athletic performance has received only limited attention. The first study to

directly assess the influence of eccentric, muscle-damaging exercise on dynamic muscle

function was undertaken by Sargeant and Dolan (1987). These authors employed an

exhaustive downhill (-25%) walking intervention to induce damage followed by

assessments of maximal short term power output using an isokinetic cycle ergometer.

Cycling at 110 rev.min-1 for 20 s, peak power was reduced by 23% 24 h after the eccentric

exercise protocol and was still 8% lower than baseline values at 96 h (Sargeant & Dolan,

1987). Decrements in MVC were of a greater magnitude than peak power, with a loss of

45% of pre eccentric exercise values at 24 h, recovering to a 30% loss at 72h (Sargeant &

Dolan, 1987).

2.6.1 Wingate 30 s cycle test

These findings have been supported by Byrne and Eston (2002b) using a 30 s Wingate test

to investigate changes in power-generating ability following EIMD. These authors reported

decrements in peak power output and isometric MVC following the performance of 100

bar-bell squats with a load corresponding to 80% concentric one repetition maximum.

There were notable differences in both the magnitude of strength and power loss and their

recovery patterns. Isometric MVC dropped by 35% immediately after the squatting

protocol and then followed a linear recovery; 26% at 24 h and 19% at 48 h. In comparison,

Wingate peak power was reduced by 13% immediately after the eccentric intervention but

dropped further, by 18% at 24 h and 16% at 48 before recovery (Byrne & Eston, 2002b).

In a more recent study, Nottle and Nosaka (2007) employed a 40 min downhill (-7%)

running damage protocol to investigate changes in Wingate peak power. In agreement with

both Sargeant and Dolan (1987) and Byrne and Eston, 2002b), greater decrements were

observed in strength than peak power immediately after downhill running (17% and 5%,

strength and peak power, respectively), yet neither strength nor power loss persisted beyond

this initial post-eccentric exercise measurement. In contrast with the two previous studies

(Sargeant & Dolan, 1987; Byrne & Eston, 2002b), recovery was rapid and peak power was

unexpectedly 5% higher than baseline measures at 120 h (Nottle & Nosaka, 2007). An

earlier investigation, Malm et al. (1999) reported no change in Wingate peak power

following a stepping protocol designed to induce muscle damage. However, the modest

increase in soreness reported by participants and the unchanged CK response indicate that

the stepping protocol employed may not have been sufficiently intense to alter muscle

function.

2.6.2 Sprint performance

Malm and colleagues (1999) also reported the changes in the performance of intermittent

cycle sprint tests (10 x 10 s all-out cycling interspersed with 50 s rest periods) which

unexpectedly improved by 8% at 48 h. No change in 30 m running sprint performance was

observed 48 h after the completion of 70 (7 x 10) drop jumps designed to induce muscle

damage (Semark et al., 1999). However, participants in the study were well-trained rugby

union and field hockey players who, although they reported moderate soreness, showed no

elevation in plasma CK levels and may have been protected from alterations to muscle

function from the drop jumps via the repeated bout effect (see section 2.5). Effective

muscle-damaging protocols such as the 100 plyometric jumps employed by Highton et al.

(2009) have been shown to result in decrements in isokinetic peak torque and increases in 5

and 10 m sprint running times at 24 h and 48 h. Twist and Eston (2005) investigated the

influence of EIMD on both cycle and running sprint performance using 100 (10 x 10)

counter-movement jumps to induce damage. Peak power output during intermittent cycle

sprinting (10 x 6 s with 24 s recovery) was reduced immediately and up to 72 h after

eccentric exercise. The rate of fatigue in the cycle tests was also reduced with the greatest

decrease observed at 48 h. Similarly intermittent sprint running time (10 x 10 m with 12 s

active recovery) increased immediately and up to 48 h after eccentric exercise (Twist &

Eston, 2005). In a more recent investigation the same authors compared the performance of

a 10 s cycle sprint and a 50 cm drop jump at 24, 48 and 72 h after completing 100 (10 x 10)

counter-movement jumps (Twist & Eston, 2007). While performances of both the cycle

sprint and the drop jump were reduced as a result of the eccentric exercise the temporal

pattern of recovery was different. Peak power output and time to reach peak power were

most severely reduced at 48 h, whereas the greatest decrement in drop jump height was

observed at 24 h (Twist & Eston, 2007). This observation led the authors to propose that

differences in the response to cycling and drop jump performance indicate that the time

course of recovery from EIMD is dependent upon the mode of dynamic exercise (Twist &

Eston, 2007).

2.6.3 Vertical jump tests

Other studies have compared performance decrements in various vertical jumps in order to

investigate the effect of muscle-damaging exercise on dynamic muscle function. Byrne and

Eston (2002a) used 100 (10 x 10) barbell squats with a load of 70% body mass to identify

differences in the performance of squat jumps, counter-movement jumps and drop jumps

following eccentric, muscle-damaging exercise. The squat jump was performed from a

squatting position before jumping vertically for maximal height whereas in both the

counter-movement and drop jumps the muscle employed the stretch-shortening cycle

(SSC). The SSC of human muscle function is the natural mode of locomotion employed in

running, walking or jumping involving the cyclical performance of a sequence of pre-

activation, active (eccentric) braking followed by concentric action (Komi, 1984). The

reductions in vertical jump performance was immediate and lasted up to 72 h, although

squat jump performance was affected to a greater extent than either counter-movement or

drop jump performance. This observation prompted the authors to propose that impairment

of muscle function in the vertical jumps was attenuated when the SSC was used (Byrne &

Eston 2002a). Similar findings have been reported by Harrison and Gaffney (2004) who

used 70 (7 x 10) maximal eccentric contractions of the knee extensors to induce muscle

damage. In contrast, when muscle damage is induced via a SSC protocol (by means of a

specially designed sledge apparatus) rather than a predominantly eccentric protocol, drop

jump performance is more profoundly affected than squat jump performance (Avela et al.,

1999; Horita et al., 1999, 2003). These authors and others (e.g. Horita et al., 1996, Nicol et

al., 2006) have also reported a ‘bi-modal’ recovery from exhaustive SSC. The bi-modal

response involves an immediate post-exercise reduction in dynamic muscle function which

is believed to result primarily from metabolic fatigue. This is followed by a partial recovery

one to two hours post-exercise and a further reduction in dynamic muscle function which

has been attributed to the inflammatory response to EIMD (Nicol et al., 2006). However,

the phenomenon of a bimodal or biphasic response may not be exclusive to damage

induced via the SSC.

MacIntyre and colleagues (1996) were the first to report a biphasic

recovery of dynamic human muscle function following 300 repetitive eccentric contractions

of the knee extensors. Furthermore, few studies report measures of muscle function

between 0 and 24 h post exercise and those that do more frequently report static force than

dynamic torque (Clarkson & Hubal, 2002).

2.6.4 Endurance exercise

The immediate and prolonged loss of power-generating ability that results from muscle

damaging exercise also impedes performance during endurance exercise. Mice have been

used to investigate running time to exhaustion and the more ecologically valid, voluntary

wheel running time, following 150 min downhill running (Carmichael et al., 2005, 2006;

Davis et al., 2007). This group of authors has reported decreases in treadmill running time

to fatigue at 24, 48 and 72h. When compared to uphill running mice, one downhill group

ran 73% less at 24 h and 69% less at 48 h (Carmichael et al., 2005). Voluntary wheel

running activity was monitored during the 12 h active dark cycles following downhill

running in all three studies. The mice spent shorter time periods on the running wheel and

travelled reduced distances in the first and second 12 h active cycles, gradually returning to

baseline activity between day 3 (Carmichael et al., 2006; Davis et al., 2007) and day 5

(Carmichael et al., 2005).

To date, only two ecologically valid studies have investigated human endurance

performance following EIMD. Marcora and Bosio (2007) reported significant differences

in self-paced 30 min time trial running performance before and 48 h after the completion of

100 (10 x 10) drop jumps. Participants ran a shorter distance at a slower speed following

EIMD. An analysis of speed, heart rate and perceived exertion variables recorded during

the run indicated that pacing strategy was not altered. In light of these findings the authors

proposed that the negative effect of EIMD on time-trial performance was mediated by the

individuals’ perception of exertion (see section 2.8.2) (Marcora & Bosio, 2007). Twist and

Eston (2009) reported similar decrements in 5 min cycle time-trial performance following

100 (10 x 10) countermovement jumps. There were significant reductions in peak power

output (-14%), mean power output (-11%), mean revolutions per minute (RPM) (-4%), and

distance covered (-4%) 48 h after EIMD but performance measures had returned to baseline

at 168 h (Twist & Eston, 2009).

2.7 Human response to dynamic exercise

Investigations reporting changes in physiological parameters during exercise following

EIMD have produced equivocal findings. The first study to document physiological

changes during exercise with EIMD was undertaken by Hamill and colleagues (1991).

Steady-state oxygen uptake ( V˙ O2 ) and heart rate (HR) data were recorded during a 15

min

run at 80% maximal V˙ O2 ( V˙ O 2 max ) before and after a 30 min bout of downhill running (at

a gradient of -15%). The V˙ O2

and HR responses of the ten recreational runners were

unchanged at 48 and 120 h after downhill running. The small, albeit significant increase in

CK activity and the development of only moderate soreness led the authors to suggest that

the downhill run may have provided insufficient stress to elicit metabolic or HR

modifications (Hamill et al., 1991). These authors also reported small but significant

decreases in hip and knee flexion during running following EIMD but overall performance

of the stride, including stride length, stride period, mechanical work, and mechanical power

was unaffected (Hamill et al., 1991).

2.7.1 Oxygen uptake

Measures of V˙ O2 during running at a fixed sub-maximal speed, such as those procured by

Hamill and colleagues (1991), provide information relating to the cost or economy of

locomotion, more commonly termed ‘running economy’. In contrast to Hamill et al.’s

(1991)findings, Braun and Dutto (2003) reported that running economy was significantly

compromised (average 3.2% increase in V˙ O2 ) during 5 min running bouts at 65, 75 and

85% V˙ O2 peak, 48 h after a 30 min downhill run (-10%) at an intensity equivalent to 70%

V˙ O2

peak.

Furthermore, these authors reported decreases in the mean stride length at all

three exercise intensities which were negatively correlated to changes in the mean energy

cost of running at all three exercise intensities (Braun & Dutto, 2003). A comparison of the

training status of the two participant groups (Hamill et al., 1991; Braun & Dutto, 2003) was

suggested as an important factor in explaining the disparate findings (Braun & Dutto,

2003). The well-trained runners in the Braun and Dutto (2003) study may have had more

refined gait patterns than the recreational runners in the Hamill et al. (1991) study. Thus

the trained runners may have been more sensitive to changes in gait resulting from muscle

damage. In support of this suggestion, Paschalis et al. (2005) reported no change in the

economy data of untrained athletes during running at approximately 55 and 75% V˙ O2 max

during the 3 days following completion of 120 (12 x 10) eccentric MVCs. Similarly,

Marcora and Bosio (2007) reported no change in the running economy of active, but not

highly trained distance runners, exercising at 70%

V˙ O 2 max for 10 min 48 h after the

completion of 100 (10 x10) drops jumps. Another study using active but not highly trained

participants also reported unaltered running economy following a series of sub-maximal

resistance exercises including barbell squat, weighted lunges and weighted step-ups (Scott

et al., 2003). It was suggested by these authors that the extent of muscle damage induced

was insufficient to produce mechanical or physiological changes that would influence V˙

(Scott et al., 2003). Chen and colleagues (2007b) have provided the only evidence of the

time course of changes in running economy following EIMD. Running economy data were

collected for five consecutive days following the completion of a 30 min downhill run (-

15%) at an intensity equivalent to 70% V˙ O2 peak (Chen et al., 2007b).

Using the same

submaximal exercise intensities as Braun and Dutto (2003) (65, 75 and 85% V˙ O2 peak)

these authors demonstrated that running economy was compromised by 4-7% for three days

after the downhill running, recovering to near baseline levels on day four (Chen et al.,

2007b). In addition, reductions in stride length (3-6%), range of motion (ROM) of the

ankle and knee joints (1-7%) and increases in stride frequency (3-7 %) were observed for

two to three days following the downhill running. These authors concluded that the time

course and magnitude of alterations in economy were more closely related to changes in

kinematic parameters than changes in muscle function as indicated by immediate and

prolonged changes in MVC (reduction of 7-21% for 4 days after downhill running) (Chen

et al., 2007b).

Measures of economy have also been assessed during cycling at a fixed sub-maximal load

and are not as susceptible to the effects of alterations in lower limb kinematics as it has

been suggested that running economy is. In fact there is consistent evidence that EIMD

does not alter cycling economy irrespective of the mode of damage employed or the

training status of the participants (Gleeson et al., 1995; Walsh et al., 2001; Moysi et al.,

2005; Schneider et al., 2007; Twist & Eston, 2009). Gleeson et al. (1995) and Schneider et

al. (2007) employed untrained participants who were required to perform bench-stepping

protocols to induce muscle damage. Participants in the other three studies were all

physically active but not highly trained and were required to perform eccentric cycling

(Walsh et al., 2001), barbell squatting (Moysi et al., 2005) and countermovement jumps

(Twist & Eston, 2009) in order to induce muscle damage. No change in V˙ O2

measures

was reported in any of the aforementioned studies during sub-maximal constant load

cycling. Furthermore, it has been reported that peak V˙ O2 measured during incremental

cycling to exhaustion was unaffected by a prior bout of eccentric, bench-stepping exercise

(Gleeson et al., 1998). The observation that cycling is not susceptible to the potentially

confounding influence of altered kinematics makes it a particularly attractive model with

which to examine the effect of EIMD on the human response to dynamic exercise. The

fixed geometry of a cycle ergometer ensures that hip and knee angles remain relatively

constant during exhaustive cycling exercise despite the development of significant localised

muscle fatigue (Dingwell et al., 2008). In contrast, reductions of up to 7% in the range of

motion of the knee joint have been reported during running following the downhill running.

(Chen et al., 2007b).

2.7.2 Oxygen uptake kinetics and muscle oxygenation

While measurements of V˙ O2 at constant sub-maximal speeds or loads provide information

relating to the energy cost of locomotion, the rate at which O2 uptake increases at exercise

onset ( V˙ O2 kinetics) can provide information pertaining to the balance of O2 delivery to

O2 utilisation in active skeletal muscle.

Schneider and colleagues (2007) were the first to

investigate the influence of EIMD on O2 uptake kinetics. Nine untrained participants each

performed square-wave transitions from unloaded to heavy intensity cycling before and 48

and 72 hours after completing 30 min of bench-stepping exercise. The heavy intensity

exercise was determined as the work load equivalent to 40% of the difference (40 %∆)

between the power output achieved at the gas exchange threshold (GET) and that achieved

at peak V˙ O2 . The phase II V˙ O2

kinetics were unaltered by the prior eccentric exercise

indicating that EIMD did not compromise oxidative function or alter the matching of O2

delivery to O2utilisation in the active muscle tissue. In addition, the unchanged slow

component indicated that the O2 cost of cycling at 40 %∆ was not altered. These findings

led the authors to speculate that they had not induced sufficiently severe muscle damage to

elicit changes in

V˙ O2

kinetics. While muscle oxygenation was not measured in this

investigation, near-infrared spectroscopy (NIRS) can facilitate the assessment of muscle

oxygenation and can therefore be used to non-invasively determine the dynamic balance

between O2

delivery and O2

utilisation.

Walsh et al. (2001) examined the kinetics of

utilisation and re-oxygenation during

ischemia and reperfusion at rest before and 2 days after 30 min of eccentric cycling.

change was reported in O2

utilisation or local O2

transport with the authors concluding

that muscle oxidative function at rest was not impaired as a result of EIMD (Walsh et al.,

2001). More recently Ahmadi et al. (2008) used NIRS to investigate O2

saturation and

desaturation kinetics at rest and during isometric contractions at 30, 50 and 80% MVC

before and after a 40 min bout of downhill walking (-25%). In contrast to the findings of

Walsh and colleagues (2001), Ahmadi et al. (2008) reported immediate and prolonged

speeding

the

NIRS-derived O2 kinetics.

The

authors

proposed

that

the

probable

mechanism for the increased O2 saturation and desaturation may have been increased O2

utilisation due to the requirements of energy demanding repair processes (Ahmadi et al.,

2008). However the authors did concede that increases in muscle blood flow subsequent to

EIMD, such as reported by Laaksonen et al. (2006), may have been responsible for the

apparent speeding of O2 saturation and desaturation kinetics (Ahmadi et al., 2008). While

muscle oxygenation kinetics have been investigated at rest and during isometric

contractions, to date, no study has attempted to investigate the influence of EIMD on

muscle oxygenation kinetics during the performance of dynamic exercise.

2.7.3 Metabolic responses to dynamic exercise

As detailed in section 2.6.4, the reduction in resting muscle glycogen uptake following

EIMD has led to speculation that increased glygogenolysis may be a consequence of EIMD

and as such may be responsible for increases in blood lactate concentration ([La]) observed

at rest (Asp et al., 1996, Asp et al., 1998) and during dynamic exercise (Braun & Dutto,

2003; Chen at al., 2007b, 2008; Gleeson et al., 1995, 1998).

Gleeson et al. (1995) were the first to report increases in immediate post exercise [La] 48 h

after a 30 min bout of bench-stepping designed to induce muscle damage. Participants

cycled for 15 min at an intensity equivalent to 80% V˙ O 2 max

48 h after either the eccentric

bench-stepping exercise or concentric uphill walking. Pre-exercise [La] was not different

between the two groups but it was higher after eccentric exercise (7.5 mmol.l-1) than after

concentric exercise (6.0 mmol.l-1). In a follow-up study, blood samples were taken before

and at 2 min intervals throughout an incremental cycle test to exhaustion and demonstrated

that following eccentric exercise [La] increased (Gleeson et al., 1998). In this investigation

the control condition was unexercised in the period prior to completion of the incremental

exercise test and demonstrated lower [La] during and 2 min after completion of the test

(Gleeson et al., 1998). These authors proposed that the higher blood [La] in the post

eccentric condition reflected a higher intramuscular [La] and an increased relative

contribution of anaerobic metabolism to energy production possibly arising from additional

recruitment of type II muscle fibres (Gleeson et al., 1998). In support of this suggestion

Braun and Dutto (2003) have reported increased blood [La] during running at 65, 75 and 85

% of peak V˙ O2 . Blood samples were taken during the 5 min rest period after each 5 min

bout and were on average 0.61 mmol.l-1 higher 48 h after the downhill running protocol.

The increase in [La], attributed to a greater reliance on glycolytic energy production,

together with altered stride mechanics were suggested as contributory factors in the

reduction in running economy (Braun & Dutto, 2003) (previously discussed in section

2.7.3). Similarly, Chen et al. (2007b) reported significant increases in [La] in blood

samples taken 3 min after the completion of each 5 min running bout at 65, 75 and 85%

V˙ O2 peak.

The authors suggested that the increases in [La] which lasted for three

days

following a 30 min downhill run may have reflected increased motor unit activation (Chen

et al., 2007b).

However, elevations in [La] may not reflect alterations in metabolic function following

EIMD.

Schneider et al. (2007) reported unchanged phase II V˙ O2 kinetics indicating that

EIMD did not compromise oxidative function (see section 2.7.3) together with elevations in

[La]. The difference between resting and end exercise blood [La] was higher following

EIMD and was attributed to an increased lactate efflux from the active muscle due to

increased membrane permeability rather than to an enhanced rate of anaerobic glycolysis

(Schneider et al., 2007). An increased lactate efflux may be countered by increased muscle

blood flow following EIMD (Laaksonen et al., 2006) which could facilitate greater [La]

clearance by improving transport to lactate metabolising muscle and other tissues.

Increased muscle blood flow may well explain for the unchanged blood [La] reported in

other studies (Hamill et al., 1991; Scott et al., 2003; Marcora & Bosio, 2007) and the

reduced blood [La] reported by Moysi et al. (2005) following EIMD.

Magnetic resonance spectroscopy (MRS) can be used to measure parameters of muscle

metabolic function including the dynamic changes in the ratio of inorganic phosphate to

phosphocreatine (Pi/PCr) and intracellular pH. Several studies have shown a significant

decrease in the resting PCr/Pi ratio following EIMD, which could be interpreted as an

increase in metabolism following the muscle injury (McCully et al., 1992; Lund et al.,

1998a, 1998b). However, due to fact that no commercially available ergometers are

capable of functioning within a MRS, only one study has investigated possible alterations

to muscle metabolism following EIMD during dynamic leg exercise inside the core of a

whole body MRS. Using a hydraulic ergometer specifically designed for quadriceps

exercise within a whole body MRS (Rodenburg et al., 1994), participants completed two

graded concentric exercise tests before and 24 h after performing stepping exercise

designed to induce EIMD (Rodenburg et al., 1995). The expected decrease in the resting

PCr/Pi ratio was observed but no differences were reported in the PCr/Pi ratio during the

exercise test. While these findings indicated that exercise metabolism was not altered by

the stepping exercise, the lack of change in several markers of muscle damage including

plasma CK activity, led the authors to conclude that muscle metabolism could feasibly be

altered by more severe EIMD (Rodenburg et al., 1995). The inconclusive nature of this one

set of findings suggests that further research employing MRS should be conducted in order

to more clearly illustrate the effect of EIMD on muscle metabolism during dynamic

exercise.

2.7.4 Ventilatory responses to dynamic exercise

The mechanisms which control ventilation during dynamic exercise are controversial but

are believed to involve elements of proportional feedback (central and carotid

chemosensory) and feed-forward (central command and muscle reflex) in varying

proportions (Ward, 2007). Below the lactate threshold, ventilation is involved in the

regulation of the arterial partial pressure of CO2 to near baseline levels. Above the lactate

threshold bicarbonate buffering of the lactic acidosis provides an important stimulus to

ventilation.

Several investigations have reported no change in ventilation following EIMD. Paschalis et

al. (2005) employed the lowest intensity exercise, with participants running at “randomly

selected velocities” of 133 and 200 m.min-1 which were equivalent to ~ 55 and ~75%

V˙ O 2 max respectively (Paschalis et al., 2005). When compared to baseline measures, no

change in ventilation was reported at either velocity for 4 days after completion of 120 (12

x 10) eccentric MVCs. Similarly, Scott et al. (2003) and Marcora and Bosio (2007) have

reported an unaltered ventilatory response to running at intensities equivalent to a blood

[La] of 2.5 mmol.l-1 (ostensibly below the lactate threshold) and 70% V˙ O ,

2 max

respectively.

Furthermore, these authors observed no influence of EIMD on blood [La]

(Scott et al., 2003; Marcora & Bosio, 2007).

Participants in the study conducted by Moysi

et al. (2005) cycled at 62% V˙ O 2 max for 6 min before and 48 h after completing a series of

muscle damaging squats. While no overall change in minute ventilation was reported the

authors did observe increases in breathing frequency which were countered by decreases in

tidal volume. An explanation for these observations and for the observation that blood [La]

was decreased by 12% at 48 was not offered (Moysi et al., 2005).

Increases in ventilation in conjunction with increases in blood [La] have been reported

during running at 65, 75 and 85% V˙ O 2 max

following EIMD (Braun & Dutto, 2003; Chen et

al. 2007b, 2008) and have been associated with impaired running economy attributed to

altered lower limb kinematics (see section 2.7.1). However, Gleeson et al. (1995) observed

increases in both ventilation and blood [La] during cycling at 80% ˙

max

48 h after

eccentric exercise. Minute ventilation was increased from 6-15 min of a 15 min bout and

was mainly due to a higher breathing frequency. Similarly, Schneider et al. (2007) reported

increases in both ventilation and blood [La] during cycling at 40%∆ (heavy intensity

exercise). While the influence of the increased lactic acidosis was considered the most

likely explanation, the increased stimulation of muscle nociceptors due to increased muscle

pain and the higher perception of effort reported were also proposed as potential

mechanisms to drive the increase in ventilation (Gleeson et al., 1995; Schneider et al.,

2007).

Schneider et al. (2007) have provided the only example of an investigation using a specific

exercise domain to study the influence of EIMD on physiological responses to dynamic

exercise. The exercise domain employed during an investigation is important in

determining the individual physiological response to dynamic exercise. Markedly different

metabolic, cardiorespiratory and perceived exertion responses are elicited from the three

sub-maximal exercise intensity domains (moderate, heavy and severe). As such, it is

important that exercise intensity is accurately defined by delineating the boundaries

between these domains when investigating these responses (Jones et al., 2009). The vast

majority of studies investigating the influence of EIMD on the performance of dynamic

exercise utilise exercise intensities determined by a percentage of maximal work capacity.

This can be rather misleading due to inter-individual differences. For example, exercise at

80% V˙ O 2

max

(e.g. Gleeson et al., 1995), may result in one individual exercising within the

heavy domain whilst another exercises within the severe domain. Future research

involving the influence of EIMD on ventilatory and other human responses to dynamic

exercise should endeavour to use specific exercise domains so that the interpretation of

findings is not confounded by individuals exercising at different relative intensities.

Increases in ventilation following EIMD may be linked to an increased lactic acidosis,

however other potential mechanisms have been investigated. In order to eliminate the

influence of metabolic control factors such as [La], Hotta and colleagues (2006)

investigated the ventilatory response to the first 20 s of dynamic knee extension exercise

following EIMD. Knee extensions with ankle weights of approximately 2.5% body mass

were repeated five to seven times. The breath by breath data were time aligned to the start

of exercise, ensemble averaged and then linearly interpolated to yield 1 s data points.

Ventilation at the onset of exercise was significantly elevated 2 and 7 days after eccentric

exercise leading the authors to suggest that alterations in the peripheral neural reflexes

contributed to the enhanced ventilatory response (Hotta et al., 2006).

2.7.5 Perception of effort during dynamic exercise

The perception of effort involves the assimilation of a number of afferent signals from

various perceptual cues emanating from different body systems including the

cardiorespiratory and neuromuscular systems which can be interpreted in both a feedback

and feed-forward manner (Hampson et al., 2001). Cues may be peripheral, produced for

example by the painful stimulation of muscle afferents following EIMD; or centrally-

derived from the cardiorespiratory responses to exercise. While the damage protocol, the

exercise mode and the exercise intensity may have a significant influence on many other

physiological responses, the weight of evidence suggests that EIMD results in an increased

sense of effort during dynamic exercise (Gleeson et al., 1995; Scott et al., 2003; Marcora &

Bosio, 2007; Chen et al., 2007b, 2008; Twist &Eston, 2009).

Gleeson et al. (1995) reported an increased sense of effort during cycling at 80% V˙ O 2 max

days after completing 30 min of bench-stepping. The higher ratings of perceived exertion

(RPE) reported were believed to reflect the weakened state of the muscles, the increase in

blood [La] and the sensation of muscle soreness (Gleeson et al., 1995). Increases in RPE

have also been reported during running following eccentric exercise (Scott et al., 2003;

Chen et al. 2007b, 2008). Scott and colleagues (2003) suggested that perceived exertion

was the best indicator of physical stress as RPE is a configuration of responses resulting

from an integration of signals, perceptions and experiences. In addition to the cues

proposed by Gleeson and colleagues (1995), altered neural control was suggested as a

possible cue to the higher perception of exertion reported following EIMD (Scott et al.,

2003). Recent studies conducted by Marcora and Bosio (2007) and Twist and Eston (2009)

have reported RPE responses during fixed-load submaximal exercise and time trial

performance. During running for 10 min at 70% V˙ O 2

max

(Marcora & Bosio, 2007) and

cycling for 5 min at 60 and then 80% of maximal power output (Twist & Eston, 2009) RPE

was higher following eccentric exercise. However, during both the running and cycling

time trials RPE was unchanged following EIMD although time trial performances were

significantly reduced. Both pairs of investigators concluded that following EIMD an

altered sense of effort mediated performance. Participants reported higher RPEs when

exercising at the same intensity and produced less force (slower running or cycling speed)

when perceiving the same effort (Marcora & Bosio, 2007; Twist and Eston, 2009).

Several studies have demonstrated alterations in the perception of force production

following eccentric exercise which persist for several days (see section 2.4.3). More recent

investigations have corroborated these findings, proposing that participants were using their

perception of the effort required to generate a given torque rather than the level of torque

itself (Carson et al., 2002; Proske et al., 2003; Weerakkody et al., 2003a; Proske et al.,

2004). These findings indicate that EIMD impairs the perception of effort or exertion rather

than the ability to match a given force as participants with EIMD with EIMD produce

reduced force for a given perceived effort and perceive higher effort when producing the

same force.

2.8 Conclusion

Direct histological analyses of muscle tissue and indirect measures of the structural and

functional status of skeletal muscle following unaccustomed, eccentric exercise are well

documented and have revealed substantial disruption. However, the effects of exercise-

induced muscle damage on the human response to dynamic exercise have been investigated

infrequently and have produced equivocal findings. Exercise modality has been found to

influence the human response following EIMD with changes observed during running more

closely related to changes in kinematic parameters than changes in muscle function.

Therefore cycling, which is not susceptible to the potentially confounding influence of

altered kinematics, is the preferred model with which to examine the effect of EIMD on the

human response to dynamic exercise. The purpose of the following studies is to investigate

the influence of EIMD on various human responses during the performance of dynamic

exercise. Specifically, investigations will focus on ventilatory and perceived exertion

responses, muscle metabolism, muscle oxygenation kinetics and pulmonary oxygen uptake.

CHAPTER 3:

COMMON METHODS

3.1 Introduction

The experiments which comprise this thesis were conducted in the exercise physiology

laboratories of the School of Sport and Health Sciences at the University of Exeter which

are accredited by the British Association of Sport and Exercise Science. In Study 3

(chapter 6) 31P-magnetic resonance spectroscopy was conducted at the Peninsula Magnetic

Resonance Research Centre at the University of Exeter. Prior to the collection of any data

all participants gave written informed consent to participate in the research, which was

approved by the School of Sport and Health Sciences Ethics Committee.

3.2 Familiarisation

Most of the participants recruited were members of the School of Sport and Health

Sciences at the University of Exeter and were familiar with the experimental testing

protocols and associated procedures. However as maximal and/or exhaustive effort was

required on most visits to the laboratory, appropriate practice was conducted. Participants

in all four studies were fully familiarised with the production of maximal voluntary

contractions using a Biodex B-2000 isokinetic dynamometer (Biodex Corp, Shirley, NY).

All participants in study 3 (chapter 6) were required to complete a familiarisation session in

a ‘mock MRI system’. During this session, participants practised single-legged, knee-

extension exercise at a rate of 40 repetitions/min in time with a visual cue projected onto

the front wall of the ‘mock’ scanner room. Vigorous encouragement was given to

participants throughout all maximal and/or exhaustive tests to ensure a maximal effort was

produced.

3.3 Eccentric muscle-damaging exercise protocol

In order to provoke muscle damage, participants in each of the four studies were required to

perform 100 squats as 10 sets of 10 repetitions using a Smith machine (Figure 3.1).

Figure 3.1 A participant being guided in the correct and safe squatting technique prior

to completion of 100 (Smith) squats.

This procedure involved the controlled, isotonic resistance of the external load of the bar,

which was calculated to correspond to approximately 70% of each participant’s body mass.

This was calculated to within 5 kg of the predicted 70% body mass as the smallest available

weights were 2.5 kg.

Prior to commencement, participants were instructed in correct and

safe lifting technique (see appendix E for risk analysis). Before the bar was disengaged it

was positioned on the participant’s shoulders and feet were positioned under the bar.

During the movement the participant’s head was kept forward, the back straight and legs

fully extended (knee = 180°). Feet were kept flat on the floor, toes pointing forward, with

equal distribution of weight through fore-foot and heel. Prior to commencement, the

appropriate foot position was marked on the floor with tape so that it could be reproduced

in each of the ten sets of ten repetitions. The descent phase involved eccentric action of the

knee extensors to lower the bar to a knee angle of just past 90°. The lifting phase involved

concentric action to return the bar to the starting position. In order to facilitate the

consistent production of 90° squats, a plastic metre rule was taped to the safety stop frame

on the guide rod using gaffer tape to provide a visual and audible guide for the participant

and investigators. When the participant achieved the required angle this could be seen in

the facing mirror and heard as the metre rule contacted the base frame of the Smith

machine.

During pilot work, a metronome was used in an attempt to control the speed of contraction

as it has been suggested that this may influence the magnitude of damage induced

(Chapman et al., 2006). However, participants struggled to maintain a set cadence of four

down to one up with the metronome set at 60 beats per minute without losing form.

Therefore this element of the protocol was abandoned and participants were encouraged to

perform controlled decent and lifting phases at their own pace whilst maintaining the

correct and safe lifting technique. After each set of ten repetitions a minimum rest period of

1 minute was allowed with participants encouraged to take as much time as they needed

between sets to ensure that the correct and safe lifting technique could be maintained.

3.4 Markers of muscle damage

In a review of measurement tools used to evaluate exercise-induced muscle damage,

Warren et al. (1999) proposed that maximal voluntary contraction (MVC) torque provided

the best measure of muscle damage resulting from eccentric contractions. These authors

were dismissive of using either changes in perceived muscle soreness or changes in the

blood levels of myofibre proteins such as Creatine Kinase (CK) as they are reported to

correlate poorly with the magnitude and time course of changes in muscle function (Warren

et al., 1999). However, we chose to include measures of soreness and plasma CK activity

in addition to those of MVC torque, in order to provide a wider physiological perspective

of the damage induced. In each study these markers of muscle damage were assessed in the

order listed below before and after completing the eccentric, muscle-damaging exercise in

order to measure the effectiveness of the protocol.

3.4.1 Creatine Kinase activity

Plasma CK activity was assessed from fingertip capillary samples. The sample was

centrifuged at 4000 RPM (2000 x g) for 5 minutes and two 20 µl samples of plasma were

then added to 1 ml of a composition of reagents supplied by Randox (CK-NAC 110,

Randox Laboratories Ltd., Crumlin, Co. Antrim, UK). Following 1 min incubation at 37˚C

and during continued incubation, absorbance at 340 nm was recorded by spectrophotometry

(Jenway 6310 spectrophotometer, Jenway, Essex, UK) at 0, 1, 2 and 3min. CK values were

calculated using the formula CK(U/l) = 8095 x Δ absorbance 340 nm/min. The mean CK

value of the two samples was calculated and used for subsequent analysis. Normal serum

values of 24-195 U/l are reported for men using this method (Szasz et al., 1976). The intra-

assay coefficient of variation for duplicate samples using this procedure in our laboratory

was 6.6 %.

3.4.2 Perceived muscle soreness

Participants assessed the soreness of their knee extensors using a blank 0–10 visual

analogue scale (VAS) (Appendix G). The VAS consisted of a 10 cm line labelled from left

(no soreness) to right (worst soreness ever). After squatting to approximately 90o knee

flexion with hands on hips, participants were asked to place a mark on the VAS to indicate

their level of soreness. Perceived pain was then quantified by measuring the distance to the

mark on the line to the nearest 0.1 cm. This procedure has been used in previous studies

(Rowlands et al., 2001; Twist and Eston, 2005, 2007).

3.4.3 Isokinetic peak torque

Following familiarisation sessions, isokinetic peak torque of the knee extensors was

measured using a Biodex B-2000 isokinetic dynamometer (Biodex Corp, Shirley, NY),

which was calibrated prior to each data collection session in accordance with the

manufacturer’s guidelines (Figure 3.2).

Figure 3.2 The assessment of isokinetic peak torque, using a Biodex B-2000 isokinetic

dynamometer (Biodex Corp, Shirley, NY)

The initialisation, system diagnostics and calibration procedures were performed

automatically by the Biodex System by pressing the Start key on the control panel.

Participants completed a standardised warm-up of 2 minutes cycling at 50 W on an

electronically braked cycle ergometer (Lode Excalibur Sport, Groningen, Netherlands)

followed

static

stretching

exercises

the

knee

extensor/flexor

muscle

groups.

Participants completed a standardised warm-up of 2 minutes cycling at 50 W on the cycle

ergometer followed

by static stretching exercises of the knee extensor/flexor muscle

groups. The participants were then seated in the isokinetic dynamometer in an upright

position with the seat angle set at 85°. The ankle was secured to the input arm of the

dynamometer on the tibia allowing full ankle motion, with the rotational axis of the

dynamometer aligned

with

the

lateral

femoral

epicondyle. All adjustments

the

dynamometer were recorded and replicated in subsequent tests.

The 80° range of motion

for each subject (from 90º to 10º knee flexion (full extension = 0°)) was manually

established by the investigator and confirmed goniometrically. The mass of the limb was

recorded by the dynamometer to enable the gravitational correction of peak torque values.

The highest of five maximal voluntary contractions (MVCs) at an angular velocity of 30

deg.s-1 was recorded. A 30-s rest period was allowed between contractions. Visual

feedback, displaying real time force and strong verbal encouragement were used to promote

maximal effort. All data were collected using the Biodex Advantage Software package and

stored on the computer for subsequent analysis. The inter-test coefficient of variation for

duplicate measures using this procedure in our laboratory was 4.9 %.

The contents of this chapter form the basis of the following publication:

Davies RC, Rowlands AV, Eston RG. Effect of exercise-induced muscle damage on

ventilatory and perceived exertion responses to moderate and severe intensity cycle

exercise. European Journal of Applied Physiology 2009; 107 (1):11-9.

CHAPTER 4:

THE EFFECT OF EXERCISE-INDUCED MUSCLE DAMAGE ON

VENTILATORY AND PERCEIVED EXERTION RESPONSES TO MODERATE

AND SEVERE INTENSITY CYCLE EXERCISE

4.1 Abstract

This study examined the effect of exercise-induced muscle damage (EIMD) on ventilatory

and perceived exertion responses to cycle exercise. Ten healthy, physically active men

cycled for six minutes at moderate intensity and to exhaustion at severe intensity before and

48 h after eccentric exercise (100 squats with a load corresponding to 70% of body mass).

Changes in ventilation and ratings of perceived exertion (RPE) were calculated for each

individual and expressed against time (moderate and severe exercise) and as a percentage

of time to exhaustion (severe exercise).

Ventilation increased during moderate exercise at

48 h ( V˙

; 34.5 ± 5.0 to 36.3 ± 3.8 L.min-1, P<0.05) but increases in RPE were not

significant.

During severe exercise at 48 h, time to exhaustion (TTE) was reduced and V˙ E

(87.1 ± 14.1 to 93.8 ± 11.7 L.min-1) and RPE (15.5 ± 1.3 to 16.1 ± 1.4) were elevated

(P<0.05). When expressed as a percentage of TTE, the differences in ventilation and RPE

values disappeared. Findings indicate that the augmented ventilatory response to cycle

exercise following EIMD may be an important cue in informing effort perception during

high intensity exercise but not during moderate intensity exercise.

4.2 Introduction

Unaccustomed exercise, particularly exercise with a high eccentric component, has a

significant impact on muscle structure and function. However, the influence of EIMD on

submaximal exercise performance remains equivocal. During submaximal running, reports

of elevated oxygen consumption ( V˙ O2 ), minute ventilation ( V˙

), respiratory

exchange ratio (RER), blood lactate concentration ([La]) and heart rate (HR) (Braun &

Dutto, 2003; Chen et al., 2007b, 2008) are countered by observations of unaltered

cardiorespiratory and metabolic responses (Hamill et al., 1991; Scott et al., 2003; Paschalis

et al., 2005; Marcora & Bosio, 2007).

During submaximal cycling, the V˙ O2 response does not appear to be altered by a prior

bout of eccentric, muscle-damaging exercise (Gleeson et al., 1995; Moysi et al., 2005;

Schneider et al., 2007; Twist & Eston, 2009). However the influence of EIMD on other

cardiorespiratory and metabolic responses remains in dispute. Elevated V˙

and HR

responses have been reported during high intensity cycling 48 h after damage-inducing,

bench-stepping and have been associated with increases in [La] (Gleeson et al., 1995;

Schneider et al., 2007). During lower intensity exercise (62% V˙ O2 max ) following squatting

exercise, V˙

, HR and [La] appear to be unchanged (Moysi et al., 2005).

Thus it would

appear that mode of damage induction and the intensity and mode of exercise are important

factors in determining the response to submaximal exercise with EIMD. However,

irrespective of the mode of damage, exercise intensity, or exercise protocol employed, the

weight of evidence supports the premise that eccentric, muscle-damaging exercise results in

an increased sense of effort (Gleeson et al., 1995; Scott et al., 2003; Marcora & Bosio,

2007; Chen et al., 2007b, 2008; Twist & Eston, 2009).

Impaired force-generation consequential to EIMD results from reduced neural input to the

muscle which serves as a protective mechanism to prevent further injury (Proske et al.,

2004). Similarly, the higher ratings of perceived exertion (RPEs) reported during sub-

maximal exercise following EIMD may also contribute to a putative centrally-mediated

protective system. The perception of effort involves the assimilation of numerous afferent

signals from a range of perceptual cues emanating from various body systems including the

cardiorespiratory and neuromuscular systems, which can be interpreted in both a feedback

and feed-forward manner (Hampson et al., 2001).

The cues which inform the perceptual response to exercise may arise from central or

peripheral sensations. Central cues, which reflect the aerobic demands of the exercise

(Åstrand & Ryhming, 1954), are derived from the cardiorespiratory system, whereas

peripheral cues include local muscle sensations (such as painful muscles) and sensations

produced by the stimulation of mechanoreceptors and chemoreceptors (Watt & Grove,

1993; Robertson & Noble, 1997). The influence of central (cardiorespiratory) cues is less

important than that of local sensations, particularly at lower exercise intensities (Mihevic,

1981; Hampson et al., 2001). Thus, the perception of exertion reported during exercise

with EIMD may be differentially influenced by central and peripheral cues dependent on

the exercise intensity.

The exercise domain employed during investigations is important in determining the

cardiorespiratory and perceived exertion responses. The three sub-maximal exercise

intensity domains (moderate, heavy and severe) elicit markedly different metabolic,

physiological and perceived exertion responses. The physiological events which separate

the different exercise domains are the lactate threshold, which marks the transition from the

moderate to heavy intensity exercise and the maximum lactate steady-state or critical

power, which marks the transition from heavy to severe intensity exercise. Thus, it is

important that exercise intensity is accurately defined by delineating the boundaries

between these domains when investigating such responses (Jones et al., 2009).

Investigations that utilise exercise intensity as determined by a percentage of maximal work

capacity can be misleading due to inter-individual differences.

For example, exercise at

80% V˙ O2 max ,

intensity

typically

used

investigate

the

influence

EIMD

submaximal exercise performance (e.g. Gleeson et al., 1995; Twist & Eston, 2009), may

result in one individual exercising within the heavy domain while another may be within

the severe domain.

Therefore, the purpose of this study was to investigate changes in ventilatory and perceived

exertion responses to cycling with and without exercise-induced muscle damage.

Specifically, to investigate these changes during 6 minutes of moderate intensity cycle

exercise and exhaustive, severe intensity cycle exercise. We hypothesised that eccentric

muscle-damaging exercise would elevate the ventilatory response to severe intensity

exercise but would not alter the response during moderate intensity exercise. In addition,

we predicted that following prior eccentric exercise the perceived exertion responses to

cycle exercise at both exercise intensities would be elevated and that the scalar time

property of perceived exertion during exercise to exhaustion would not be affected.

4.3 Method

Participants

Ten healthy, physically active men volunteered to participate in the study. All were

asymptomatic of illness and pre-existing injuries and had not participated in any resistance

training of the lower limbs for at least six months prior to assessment. Their characteristics

are shown in Table 4.1. Participants gave written informed consent to participate in the

research, which was approved by the Ethics Committee of the School of Sport and Health

Sciences at the University of Exeter (See appendices B, C and D for exemplar participant

information sheet, participant consent form and ethical approval certificate).

Table 4.1 Participant characteristics (N = 10).

Characteristic Mean (± SD)

Age (yrs) 21.5 (± 1.2)

Height (m) 1.82 (± 0.06)

Mass (kg) 79.8 (± 8.2)

Max V˙ O2 (ml kg min )

. -1 -1 48 (± 3)

Max HR (beats.min-1) 194 (± 6)

Max WR (W) 353 (± 30)

WR at GET (W) 120 (± 14)

Moderate WR (W) 96 (± 11)

Severe WR (W) 283 (± 24)

Maximal (max), gas exchange threshold (GET) and other work rate (WR) values are those measured during

the ramp test.

Participants were requested not to take any anti-inflammatory drugs for the duration of the

study and were instructed to report to the laboratory in a rested state, having completed no

strenuous exercise in the preceding 24 h. Changes in V˙ O2 kinetics and

deoxyhaemoglobin

kinetics due to EIMD were assessed in a sub-sample and this data has been reported

elsewhere (Chapter 7).

Procedures

Exercise testing

Participants were required to visit the laboratory at the same time of day (± 1 h) on four

occasions over a 3-week period. All testing was performed on an electronically braked

cycle ergometer (Lode Excalibur Sport, Groningen, Netherlands). During the first visit,

after measurement of height and body mass (SECA, Hamburg, Germany) participants

completed an incremental (ramp) cycle exercise test to exhaustion to establish maximal

oxygen uptake ( V˙ O2 max ) and gas-exchange threshold (GET) and to establish future work

intensities (Table 4.1). Participants cycled at a self-selected pedal rate (between 70 and 90

rpm) and this pedal rate along with the saddle and handlebar height and configuration were

recorded and reproduced in subsequent tests. The ramp test consisted of 4 minutes of

baseline cycling at 0 W followed by a continuous increase in work rate of 1 W every 2 s

(i.e. 30 W.min−1) until the subject was unable to continue.

The V˙ O was identified as

2 max

the highest 30-s mean value recorded prior to the participant’s volitional termination of the

test. The GET was determined independently by two experienced reviewers using the V-

slope method (Beaver et al., 1986).

This involves visual inspection of individual plots of

V˙ CO2

against V˙ O2

to establish the first disproportionate increase in V˙ CO2 . The work

rates that required 80% of the GET (moderate exercise) and 70% of the difference ()

between the GET and V˙ O2

max

(severe exercise) were calculated, with account taken of the

mean response time of the V˙ O2 adaptation to ramp exercise (approximately 2/3 of the

ramp rate i.e. minus 20 W) (Whipp et al., 1981). On the second and fourth visits, before and

48 h after eccentric exercise, respectively, participants cycled at a self-selected pedal rate

(between 70 and 90 rpm) for six min at a constant work rate (WR) of 80% GET (moderate).

After six min rest, participants cycled at a constant work rate of 70% (severe intensity)

and continued until volitional termination of the test at exhaustion. Participants received

strong verbal encouragement to continue exercising for as long as possible in all exhaustive

tests.

Eccentric exercise

On the third visit participants completed the eccentric, muscle-damaging exercise protocol

which comprised 100 (Smith) squats performed as 10 sets of 10 repetitions. The load on the

bar was calculated to correspond to 70% of each participant’s body mass. For further

details of these procedures please refer to Chapter 3.

Measurements

Markers of muscle damage

Markers of muscle damage (muscle soreness and isokinetic peak torque) were measured

immediately before and then 30 minutes and 48 hours after eccentric exercise. In addition

plasma creatine kinase (CK) activity was assessed immediately before and then 30 minutes,

24 and 48 hours after eccentric exercise. Due to availability at 24 h, only 8 participants

were measured for CK activity.

Perceived soreness of the knee extensors was assessed using a blank 0–10 visual analogue

scale (VAS). The VAS consisted of a 10 cm line labelled from left (no soreness) to right

(worst soreness ever). Participants squatted to 90o knee flexion with hands on hips and then

placed a mark on the VAS to indicate their level of soreness. Perceived pain was then

quantified by measuring the distance from 0 to the mark on the line to the nearest 0.1 cm.

Plasma CK activity was assessed from fingertip capillary samples. The sample was

centrifuged at 4000 RPM (2000 x g) for 5 minutes and two 20 µl samples of plasma were

then added to 1 ml of reagents (Randox CK-NAC 110, Randox Laboratories Ltd., Crumlin,

Co. Antrim, UK). The solution was then incubated at 37˚C and absorbance at 340 nm was

recorded by spectrophotometry (Jenway 6310 spectrophotometer, Jenway, Essex, UK) at 1,

2 3 and 4 min. CK values were calculated using the formula CK(U/l) = 8095 x Δ

absorbance 340 nm/min. The mean CK value of the two samples was calculated and used

for subsequent analysis. Normal serum values of 24-195 U/l are reported for men using this

method (Szasz, 1976). The intra-assay coefficient of variation for duplicate samples using

this procedure in our laboratory was 6.6 %.

Isokinetic peak torque was measured using a Biodex B-2000 isokinetic dynamometer

(Biodex Corp, Shirley, NY), which was calibrated prior to each data collection session in

accordance with the manufacturer’s guidelines. Following familiarization sessions,

participants performed five maximal voluntary contractions (MVCs) at 30 deg·s-1 with a

rest period of 30 s between contractions. The inter-test coefficient of variation for duplicate

measures using this procedure in our laboratory was 4.9 %.

Visual feedback, displaying

real time force, was used to encourage maximal effort. For further details of these

procedures please refer to Chapter 3.

Exercise test measures

Pulmonary gas exchange was measured breath-by-breath via an online gas analysis system

(Cortex MetaMax 3B, Biophysik, Leipzig, Germany) throughout all exercise tests. Changes

in breathing frequency (fR), minute ventilation ( V˙

), oxygen uptake ( V˙ O2 ) and V˙

/ V˙

were recorded continuously during the tests via the Cortex Metasoft 3.1 software. The

system was calibrated prior to every test in accordance with manufacturer’s guidelines

against known concentrations of cylinder gases (15% oxygen, 5% carbon dioxide) and a 3-l

calibration syringe (for flow volume). Heart rate (HR) was monitored using a wireless

chest strap telemetry system (Polar Electro T31, Kempele, Finland) and recorded

continuously via a link to the Cortex gas analysis system in all exercise tests.

Participants were familiarised with Borg’s 6-20 Rating of Perceived Exertion (RPE) Scale

and provided with standardised instructions on how to employ the scale (Borg, 1998).

Participants were encouraged to focus on their overall perception of exertion when

reporting their RPE, which were recorded during the last 15 seconds of each minute of all

exercise tests (Figure 4.1).

The blood lactate response was assessed from fingertip blood samples collected

immediately before and immediately after the moderate and severe intensity exercise bouts.

Samples were collected into a Lithium heparin microvette (CB300, Sarstedt AG & Co.,

Nümbrecht, Germany) and were subsequently analysed for blood lactate concentration

using a YSI 2300 STAT plus analyzer (Yellow Springs, Ohio, USA).

Figure 4.1

RPE Scale. A participant reporting his RPE during cycle exercise using the Borg 6-20

Statistical analysis

Markers of muscle damage

Changes in the markers of muscle damage (isokinetic peak torque, perceived muscle

soreness and creatine kinase activity) were analysed using a series of one way repeated

measures analyses of variance (ANOVAs).

Data were initially checked for assumptions of

normality and as the CK activity data were not normally distributed; these values were log-

transformed prior to statistical analysis. Following transformation CK activity data were

normally distributed (see appendix H).

Exercise test measures

Changes in fR,V˙

V˙ O2 , HR, V˙

/ V˙ O2 and RPE were calculated for each individual and

expressed against time (minute values) for both moderate and severe intensity exercise, and

in the case of severe exercise, as a % of time to exhaustion (% time). These values were

analysed via a series of two-factor (test x time) fully repeated measures ANOVAs. Where

assumptions of sphericity were violated (P <.05) the Greenhouse-Geisser (GG) correction

factor was applied to adjust the degrees of freedom. Where a significant test x time

interaction was observed, post hoc Tukey tests modified for repeated measures (Stevens,

2002) were run to determine where significant differences occurred. Paired t-tests were

used to determine significant differences in blood lactate concentration [La] and time to

exhaustion during severe intensity exercise pre- and post-eccentric exercise. All data were

analysed using the statistical software package SPSS for Windows (version 13). Statistical

significance was set at 0.05. A P-value of between 0.05 and 0.10 was considered a trend.

4.4 Results

Markers of muscle damage

The eccentric exercise was effective in provoking significant changes in all markers of

muscle damage. Table 4.2 shows changes in isokinetic peak torque, muscle soreness and

CK activity before and after eccentric exercise. Isokinetic peak torque (30 deg.s-1)

decreased to 86% of pre damage values at 30 min recovering to 89% at 48h (F (2, 18) = 13.37

P < 0.001). Significant soreness was reported 30 min after eccentric exercise with the

highest values reported at 48 h (F (2, 18) = 71.49 P < 0.001). CK activity increased after

eccentric exercise, with the highest values observed at 24 h (FGG (1.4, 9.8) = 13.13 P < 0.05).

Table 4.2 Changes in markers of muscle damage. Mean (± SD) values before (pre) and

30min and 48h after eccentric exercise

Measured variable pre 30 min 24 h 48 h

CK activity (U/l) 178 ±129 244 ± 181 794 ± 690* 88 ± 217

Soreness 0.5 (0.5) 5.4 (1.6)* n/a 6.8 (1.6)*

Peak Torque (Nm) 30 deg.s-1 279(45) 239 (28)* n/a 248 (35)*

N = 10, Soreness and Peak Torque; N = 8, CK activity* Significantly different from pre value (P < 0.05).

Exercise test measures

Moderate intensity exercise

Table 4.3 shows mean (± SD) changes fR, V˙E , V˙ O2 , HR, V˙E / V˙ O2 and RPE respectively

during cycling at moderate intensity (80% GET) pre and 48 h post eccentric exercise. Main

effects for test for fR (F (1, 9) = 7.29, P = 0.024), V˙

(F (1, 9) = 6.36, P = 0.033) and HR (F

(1,

9) = 12.52, P = 0.006) indicated an increase in values following eccentric exercise.

There

were no significant differences in V˙ O2 , V˙E / V˙ O2 or RPE as a result of eccentric exercise

(P > 0.05). Similarly, there were no significant differences in pre and post exercise blood

lactate concentration before and after eccentric exercise (pre: 1.13 ± 0.43, 1.08 ± 0.29 and

post: 0.98 ± 0.21, 1.10 ± 0.28 mmol.l-1 before and after eccentric exercise, respectively) (P

> 0.05).

Table 4.3 Changes in fR, V˙E , V˙ O2 ,HR, V˙E / V˙ O2 and RPE during moderate

intensity exercise. Mean (± SD) values before (pre) and 48h after (post) eccentric exercise.

V˙ E

V˙

HR V˙E /

V˙ O2

RPE

(breaths.min-1) (L.min-1) (ml.kg-1.min-1) (Beats.min-1)

Pre 20.6 (± 3.5) 34.5 (± 5.0) 19.9 (± 0.9) 109 (± 7) 21.8 (± 1.6) 9.9 (± 1.7)

Post 21.8 (± 3.2)* 36.3 (± 3.8)* 20.1 (± 1.8) 116 (± 8)* 22.7 (± 1.2) 10.6 (± 1.6)

* Significantly different from pre value (P < 0.05) N = 10

Severe intensity exercise to exhaustion

As expected, time to exhaustion was significantly reduced following eccentric exercise

(7.33 ± 2.26, 6.37 ± 2.16 min pre- and post-eccentric exercise, respectively) (t (9) 2.64, P =

0.027). Before eccentric exercise all 10 participants completed a minimum of 5 min severe

intensity. However, 48 h after eccentric exercise the minimum time achieved by all

participants was reduced to 4 min. Pre-exercise [La] was unchanged, but end-exercise [La]

was significantly lower following eccentric exercise (t (9) = 3.31, P =0.009) (pre-exercise:

0.90 ± 0.34, 0.79 ± 0.34 and end exercise: 9.33 ± 1.73, 8.45 ± 1.51 mmol.l-1 before and

after eccentric exercise, respectively).

Minute-by-minute measures

In order to investigate minute-by-minute changes in fR, V˙E , V˙ O2 ,HR, V˙E / V˙ O2 and RPE,

the first 4 minutes of data were analysed. Figure 4.2 a–f shows minute-by-minute changes

for minutes 1-4 in fR, V˙

, V˙ O2 , HR, V˙

/ V˙ O2

and RPE respectively during cycling at

70%∆ pre and post eccentric exercise. A main effect for time was observed for all

variables (P < 0.05).

Main effects for test for fR (F (1, 9) = 10.53, P = 0.010), V˙

(F (1, 9) =

9.50, P = 0.013), V˙E / V˙ O2 (F (1, 9) = 8.47, P = 0.017), and RPE (F (1, 9) = 8.15, P = 0.019)

and a trend for HR (F (1, 9) = 4.43, P = 0.065) showed that values had increased following

eccentric exercise. There was an interaction (test*time) for fR (FGG (1.3, 11.3) = 5.25, P =

0.036) and V˙E (F (3, 27) = 3.19, P = .039). Post hoc tests indicated that following eccentric

exercise, fR and V˙E were significantly higher for all but the first minute of exercise. There

were no significant differences in V˙ O2 (P > 0.05).

Percentage time values

Figure 4.3 a–f shows % time changes in fR, V˙E , V˙ O2 , HR, V˙E / V˙ O2 and RPE respectively

during cycling at 70%∆ pre and post eccentric exercise. When expressed as a % of time to

volitional exhaustion, the main effects for time remained but the main effects for test for fR,

V˙E , HR and RPE disappeared. However, there was a main effect for test for V˙ O2 (F (1,

9) = 5.25, P = 0.048) which showed that values had decreased following eccentric

exercise. This was accompanied by an increase in V˙E / V˙ O2 (main effect, F (1, 9) = 5.17,

P = 0.049).

Figure 4.2

Minute values (severe intensity

exercise). Minute-by-minute

changes in a: breathing frequency

(fR), b: minute ventilation ( V˙

c: oxygen uptake ( V˙ O2 ), d:

HR, e: V˙E / V˙ O2 and f: RPE

during cycling at 70%∆ pre- and

48h post- eccentric exercise.

Values are mean (± SEM).

Significant main effect for time for

all measures (P < 0.05).

† Significant main effect for test (P

< 0.05).

‡ Significant interaction effect

(test*time) (P < 0.05).

* Significantly different from pre

value (P < 0.05).

Figure 4.3

Percentage time values (severe

intensity exercise). Changes in a:

breathing frequency (fR), b: minute

ventilation ( V˙E ), c: oxygen

uptake

( V˙ O2 ), d: HR, e: V˙E /

V˙ O2

and f:

RPE during cycling at 70%∆ pre-

and 48h post-eccentric exercise set

against % time to volitional

exhaustion.

Values are mean (± SEM).

Significant effect for time for all

measures (P < 0.05).

† Significant main effect for test

(P < 0.05).

‡ Significant interaction effect

(test*time) (P < 0.05).

* Significantly different from pre

value (P < 0.05)

4.5 Discussion

This investigation demonstrates that a prior bout of eccentric, muscle-damaging exercise

augments the ventilatory response not only during severe intensity (70% ∆) cycle exercise

but also during moderate intensity (80% GET) cycle exercise. In addition, this study shows

that the perception of exertion is elevated during severe intensity exercise but appears to be

unaffected by a prior bout of eccentric exercise during moderate exercise.

Markers of muscle damage

As anticipated, the squatting protocol was effective in provoking changes in the markers of

muscle damage in all participants. The significant decreases in peak torque observed

following eccentric exercise concur with previously reported findings (Byrne et al., 2001).

Perceived muscle soreness was elevated above baseline measures at 30 min after the

eccentric exercise with the highest values reported at 48 h. These findings are consistent

with the previously reported characteristic temporal profile of increased muscle soreness

following eccentrically-biased exercise (e.g. Twist & Eston, 2005). As expected, the time

course of the CK response did not reflect the alterations in muscle function or perceived

soreness (Warren et al., 1999). However, the CK efflux, which was greatest at 24 h, does

provide indirect evidence of increased myocyte membrane permeability (Allen et al., 1995).

A rapid rise and peak in CK activity similar to that observed herein has previously been

observed following a similar eccentric exercise protocol (100 squats @ 70% body mass,

Byrne & Eston, 2002a).

Exercise test measures

The elevated ventilatory response to severe intensity cycle exercise observed in this study

concurs with previously reported observations during high-intensity, fixed-load cycle

exercise following muscle-damaging exercise (Gleeson et al., 1995; Schneider et al., 2007).

However, the observation that V˙

and fR were also increased during moderate intensity

exercise (<GET) was unexpected. Using a similar squatting protocol, which produced

comparable levels of strength loss (-14% at 48 h) and an exercise intensity of 62% V˙ O2max

Moysi et al. (2005) reported no change in ventilation or HR. In contrast, increases in both

ventilation and HR were observed in the present study, despite the fact that subjects

exercised a considerably lower intensity, 80%GET, equivalent to ~27% V˙ O2max for these

individuals.

The elevated ventilatory response observed whilst exercising with EIMD or DOMS has

been associated with alterations in metabolic factors. Specifically, it has been assumed that

an increase in [La] contributes to an augmented ventilatory response. The elevated [La]

may result from an increased dependence on type II fibres and a corresponding shift to

increased glycolytic energy production following muscle-damaging exercise (Braun &

Dutto, 2003; Chen et al., 2007b; Gleeson et al., 1995). However, no such change in [La]

was observed in the current study at either exercise intensity. Importantly, the observation

that the ventilatory response following EIMD is higher during exercise below GET

suggests that the altered exercise response may not be due to changes in metabolic factors.

In support of this proposition, Schneider et al. (2007) observed that phase II V˙ O2 kinetics

were not altered by DOMS and concluded that elevated [La] did not result from altered

oxidative function. Rather, these authors suggested that higher levels of [La] arose from an

increased rate of lactate efflux from damaged myocytes due to increased membrane

permeability. However, the higher rate of lactate efflux may be countered by enhanced

clearance

facilitated

increased

muscle

blood

flow

(Laaksonen

al.,

2006)

and

accordingly elicit an unchanged [La] response. Whilst the putative influence of [La] on the

ventilatory response should not be dismissed; in light of the findings of this study, other

potential stimuli demand consideration.

The increases in V

and fR observed in the present study during moderate and severe

intensities and in V˙E / V˙ O2 during severe intensity may be attributed to alterations in

neural factors. The complex mechanisms involved in ventilatory control during exercise

involve a combination of central command and afferent feedback but these are poorly

understood. However, ventilation is known to increase in response to painful stimulation

(Haouzi et al., 2004). Thus the local muscle pain which occurs as a result of eccentric

exercise, such as observed in the present study, would be expected to have a stimulatory

effect on ventilation. It has been proposed that group III and IV afferent fibres located in

and around the blood vessels of exercising muscle are involved in modulating the ventilatory

response (Haouzi et al., 2004). Distension of these blood vessels, such as the changes

in the capillary lumen shape observed by Kano et al. (2005) following eccentric exercise,

would provoke a discharge from the afferent fibres leading to an increase in ventilation.

Thus, neural monitoring of peripheral vascular and local muscular events may, in part,

account for the augmented ventilatory response observed. Hotta et al. (2006) suggested

that changes in neural factors contribute not only to an enhanced ventilatory response but

also to alterations in force generation. Following eccentric exercise increased motor unit

activation may be necessary in order to achieve a given sub-maximal force (Semmler et al.,

2007). Similarly, a greater sense of effort is reported when producing a specific force

following eccentric exercise (Proske et al., 2004).

Consistent with these observations, subjects in the present study reported higher ratings of

perceived exertion (RPE) during fixed-load, severe intensity cycle exercise following

eccentric exercise, although RPE appeared to be unchanged during moderate exercise.

Jameson and Ring (2000) have suggested that during cycle exercise, ratings of perceived

exertion are based on a combination of leg muscle pain and feelings of breathlessness.

Thus the increased leg muscle soreness experienced by participants in this study following

the eccentric exercise is likely to have provided an important peripheral cue to inform the

RPE response. Similarly, the increased ventilatory response may have provided an

important central cue. Although the relative contribution of various central and peripheral

cues is poorly understood, central cues may be less important than peripheral cues,

particularly during low intensity exercise (Mihevic, 1981; Hampson et al., 2001). It is of

interest to note that the increased muscle pain and the elevated ventilatory response

experienced by participants in the present study at 48h did not significantly influence the

perception of exertion during moderate intensity exercise. However, during severe intensity

exercise, where the central, ventilatory cues may play a more influential role in informing

the perception of exertion, RPE was elevated.

The higher perception of exertion reported in the severe exercise bout at 48h may account

for the reduction in time to end exercise. Elevated RPE has previously been associated

with reduced time-trial performance in running (Marcora & Bosio, 2007) and in cycling

(Twist & Eston, 2009). Furthermore, the amplified ventilatory response may contribute to

the premature termination of exercise. Acute respiratory muscle fatigue may have

increased the severity of locomotor muscle fatigue via a respiratory muscle metaboreflex

increase effort perception and further centrally-mediated reductions in motor output (Romer

& Polkey, 2008).

The complex interplay of central and peripheral fatigue factors is of great importance in

determining the duration of an individual participant’s exercise performance. However, the

participant’s decision to terminate exercise is ultimately a conscious behavior based on the

perception of alterations in sub-conscious homeostatic control systems (St Clair Gibson et

al., 2003). As such, the perception of exertion may be considered fundamental to the

individual exercise response when a participant is required to exercise to ‘volitional

exhaustion’. Pertinent to this is the observation that minute-by-minute differences in

perceived exertion values reported during severe exercise before and after eccentric

exercise are eliminated when expressed as a percentage of total exercise duration. Thus,

further evidence is provided to support the proposition that there is a scalar-linear

relationship between the rating of perceived exertion and exercise duration (Eston et al.,

2007, Crewe et al., 2008; Faulkner et al., 2008; Joseph et al., 2008). Furthermore, the

observation that the differences in V

and fR are also eliminated when expressed as a

proportion of time to exhaustion may provide some insight into the cues that inform the

perceived exertion response when cycling at high-intensity after a prior bout of eccentric

exercise.

4.6 Conclusion

This is the first study to investigate the influence of muscle-damaging exercise on the

ventilatory and perceived exertion responses to cycling in specific exercise domains above

and below the gas exchange threshold. Findings suggest that there is a strong link between

the augmented ventilatory response to cycling following eccentric exercise and the higher

rating of perceived exertion reported during exercise above GET. Furthermore additional

evidence is provided to support the observation that perceived exertion scales with exercise

duration during exercise to exhaustion.

The contents of this chapter are currently under review for publication:

Davies RC, Rowlands AV, Poole DC, Jones AM and Eston RG. Exercise-induced

muscle damage dissociates the Lactate and Gas exchange thresholds. Currently under

review.

CHAPTER 5:

THE EFFECT OF ECCENTRIC EXERCISE-INDUCED MUSCLE DAMAGE

ON THE GAS EXCHANGE THRESHOLD

5.1 Abstract

We tested the hypothesis that exercise-induced muscle damage (EIMD) would increase the

ventilatory ( V˙ E ) response to incremental/ramp cycle exercise (lower the gas exchange

threshold), without altering the blood lactate profile thereby dissociating the gas exchange

and lactate thresholds. It was considered that this intervention might provide a broader

understanding of the mechanisms underlying the relationship between the GET and lactate

threshold (Tlac). Ten physically active men completed maximal incremental cycle tests

before (pre) and 48 h after (post) performing eccentric exercise comprising 100 squats with

a load corresponding to 70% body mass. Pulmonary gas exchange was measured breath-

by-breath and finger-tip blood sampled at 1-min intervals for blood [La] determination.

GET occurred at a lower work rate (pre, 136 ± 27 W; post, 105 ± 19 W, P< 0.05) and V˙

(pre, 1.58 + 0.26; post, 1.41 + 0.14 l.min-1, P< 0.05) after eccentric exercise. However,

Tlac occurred at a similar work rate (pre, 161 ± 19 W; post, 158 ± 22 W, P> 0.05) and

V˙ O2

(pre, 1.90 ± 0.20 l.min-1; post, 1.88 ± 0.15 l.min-1, P > 0.05) after eccentric exercise.

These findings demonstrate that EIMD dissociates the V˙ Eresponse to incremental/ramp

exercise from the [La] response indicating that V˙ Emay be controlled by additional or

altered neurogenic stimuli following eccentric exercise. Thus, due consideration of prior

eccentric exercise should be made when using the gas exchange threshold to provide a non-

invasive estimation of the lactate threshold.

5.2 Introduction

During dynamic incremental or ramp exercise protocols expired carbon dioxide production

( V˙ CO2 ) increases disproportionately as a function of V˙ O2 above what has been termed

the gas exchange threshold (GET) (Beaver et al., 1986). Traditionally, the GET has been

considered to result from an obligatory increase in non-metabolic CO2 production (and

associated increases of ventilation, ( V˙ E ) due principally to plasma bicarbonate buffering

of lactic acid-derived H+ (Beaver et al., 1986; Wasserman et al., 1990). Thus, identification

of the GET provides the basis for the non-invasive estimation of the lactate threshold (Tlac)

(Beaver et al., 1986; Caiozzo et al., 1982; Wasserman et al., 1973, 1990). However, the

V˙ E and

lactate

responses

incremental

exercise

have

been

dissociated

various

experimental and clinical conditions including exercise training (Poole & Gaesser, 1985),

glycogen depletion (Hughes et al., 1982; Sabapathy et al., 2006) and McArdle’s disease

(Hagberg et al., 1982; Paterson et al., 1990; Riley et al., 1993). In contrast, in a recent

study dichloroacetate (DCA) was reported to reduce both blood lactate [La] and V˙ E

during

incremental exercise thereby supporting the existence of a causal link between bicarbonate

buffering of lactic acidosis and increases in

V˙ E (Wilkerson et al., 2009). Today it is

appreciated that a wide variety of humoral and neural control mechanisms (central

command, afferent feedback from carotid body chemoreceptors and from contracting

muscles) contribute to the

V˙ E response to incremental/ramp exercise (Dempsey et al.,

2006; Haouzi, 2006; Haouzi et al., 2004; Waldrop & Iwamoto, 2006). While a complex

integration of these mechanisms is understood to mediate the V˙ E

response, it is likely that

under different conditions the proportional contribution of each of these might change.

During rhythmic exercise, group III and IV afferents are known to provide some V˙ E

drive

via mechanical stimuli such as local muscular distortion, vascular distension and increased

intramuscular pressure as well as via local humoral mediators including H+, lactate and

bradykinin (Ward, 2000; Whipp et al., 1981). Eccentric exercise causes profound muscle

structural and functional perturbations collectively referred to as exercise-induced muscle

damage (EIMD) (Clarkson et al., 1992). EIMD augments discharge from group III and IV

afferents (Avela et al., 1999; Komi, 2000; Taguchi et al., 2005) and this, rather than

elevated blood lactate concentration which is not an obligatory consequence of EIMD

(Twist & Eston, 2009), most probably contributes to the greater V˙ E

response to constant-

load cycle exercise evoked by EIMD (Gleeson et al., 1995; Schneider et al., 2007; Twist &

Eston, 2009) (Chapters 4).

The influence of a prior bout of eccentric exercise and the resultant EIMD on the V˙ E

and

gas exchange responses to incremental exercise has received very limited attention. Only

one previous study examined physiological responses to maximal incremental cycling;

unfortunately the bench-stepping protocol utilised in that investigation produced only mild

soreness (Gleeson et al., 1998). Thus, that bench-stepping protocol (Gleeson et al., 1998)

may not have increased the discharge from group III and IV afferents as much as a

procedure designed specifically to induce more severe EIMD. To our knowledge, the

influence of eccentric muscle-damaging exercise on the GET has not been investigated

previously. It is our contention that an eccentric exercise protocol such as that utilised

previously in our laboratory (Chapter 4) is likely to exacerbate the V˙ E response to

incremental/ramp exercise and, in so doing, potentially dissociate the V˙ E , gas exchange

and lactate responses.

It was the purpose of this investigation to determine the influence of EIMD on the GET and

Tlac responses determined during ramp exercise. Specifically, we tested the hypothesis

that completion of 100 squats (performed as 10 sets of 10 reps at ~ 70% body mass) would

augment the V˙ E

response (lower the GET) to subsequent incremental/ramp exercise in the

absence of an altered blood lactate profile thereby dissociating the GET from the lactate

threshold. It was speculated that this intervention might provide mechanistic insights into

the relationship between lactate accumulation and ventilatory control in the presence of

overt muscle damage.

5.3 Methods

Subjects

Ten healthy, physically active male subjects (age, 25 ± 7 years; mass, 80.1 ±9.9 kg; height,

1.80 ± 0.08 m) volunteered to participate in this study. All participants were asymptomatic

of illness and pre-existing injuries and had not performed any resistance training of the

lower limbs within the previous 6 months. Participants provided written informed consent

to participate in the study which was approved by the Ethics Committee of The School of

Sport and Health Sciences at The University of Exeter (See appendices B, C and D for

exemplar participant information sheet, participant consent form and ethical approval

certificate).

Experimental Design

Ramp incremental exercise test

Participants performed two ramp incremental exercise tests to volitional exhaustion on an

electronically-braked cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands)

(Figure 5.1). Each participant’s preferred seat height and handlebar positions were

recorded during their first test and replicated in the subsequent test. The height and mass

(SECA, UK) of each participant was also recorded. Exercise tests were performed at the

same time of day ± 1 h for each participant, before and 48 h after performing eccentric,

muscle-damaging exercise. Following 3 min of unloaded baseline cycling, the work rate

was increased in a ramp fashion by 1 W every 2 s (30 W.min−1) until the subject was

unable to continue despite receiving strong verbal encouragement. The participants were

required to maintain a pedal rate of 80 rpm during both incremental tests.

Figure 5.1 A participant performing a ramp incremental cycle exercise test.

Eccentric, muscle-damaging exercise protocol

Participants completed 100 (Smith) squats, performed as 10 sets of 10 repetitions with the

load on the bar corresponding to ~70% of each participant’s body mass. For further details

of this procedure please refer to Chapter 3.

Measurements

Assessment of muscle damage

All indicators of muscle damage (perceived muscle soreness (using a 0-10 visual analogue

scale (VAS)), creatine kinase (CK) activity and isokinetic peak torque (30 deg.s-1)) were

measured in the order listed, immediately before, 30 minutes after and 24 and 48 h after

performing the eccentric, muscle-damaging exercise protocol. For further details of these

procedures please refer to Chapter 3.

Ramp incremental exercise

Throughout the cycle exercise tests, before and 48 h after the muscle-damaging protocol,

pulmonary gas exchange was measured breath-by-breath via an online gas analysis system

(Cortex MetaLyzer 3B, Biophysik, Leipzig, Germany). Participants wore a nose clip and

breathed through a low-dead-space, low-resistance mouthpiece. Gas exchange was

measured throughout the tests using the Cortex Metasoft 3.1 software. The system was

calibrated prior to every test in accordance with manufacturer’s guidelines against known

concentrations of cylinder gases (15% oxygen, 5% carbon dioxide) and a three litre

calibration syringe (Hans Rudolph, Kansas City, USA) for gas flow. Inter-test coefficients

of variation for V˙

E ,

V˙

and V˙

CO2

responses using this equipment in our laboratory

were 4.1%, 3.1% and 4.2% (for V˙ E , V˙ O2 and V˙ CO2 , respectively).

The gas exchange data sets were blind reviewed in order to determine the V˙ O2 peak and gas

exchange threshold (GET).

The V˙ O2 peak was determined as the highest 30-s average value

recorded before the participant’s volitional termination of the test. The GET was

determined from a cluster of measures which included 1) the first disproportionate increase

in CO2 production ( V˙ CO2 ) from visual inspection of individual plots of V˙ CO2 versus

V˙ O2 (V-slope method (Beaver et al., 1986)) and 2) an increase in the ventilatory

equivalent for O 2

( V˙ E / V˙ O2 ) with no concomitant increase in the ventilatory equivalent

for CO2 ( V˙ E / V˙ CO2 ) (Caiozzo et al., 1982).

Heart rate was monitored continuously using a wireless chest strap telemetry system (Polar

Electro T31, Kempele, Finland) and was recorded via a link to the Cortex gas analysis

system. Finger-tip blood samples were collected into a capillary tube immediately before,

after and at 1-min intervals during each incremental exercise test. Samples were

subsequently analysed for whole blood lactate (YSI 2300 Sport, Yellow Springs, Ohio,

USA). The blood lactate concentration [La] data sets were blind reviewed in order to

determine the lactate threshold (Tlac). Tlac was determined as the V˙ O2 associated with

the work rate prior to the first clear and sustained increase in [La] above resting levels from

visual inspection of individual plots of [La] vs. V˙ O2 .

Subjects were also asked to report their rating of perceived exertion (RPE) at 1-min

intervals throughout the incremental tests.

All subjects were familiarized with the Borg 6-

20 RPE scale and provided with standardised instructions on how to employ the scale prior

to testing (see Appendix F).

Statistical analysis

Indicators of muscle damage

Changes in the indicators of muscle damage (perceived muscle soreness, creatine kinase

activity and isokinetic peak torque) were analysed using a series of one-way repeated

measures analyses of variance (ANOVAs). Post-hoc Tukey tests modified for repeated

measures (Stevens, 2002) were run to determine where significant differences occurred. As

the CK activity data were not normally distributed these values were log-transformed prior

to statistical analysis (Twist & Eston, 2005) (see appendix H).

Ramp Incremental Exercise

Paired t-tests were used to determine significant differences in time to exhaustion, peak

values and gas exchange threshold (GET) and lactate threshold (Tlac) values pre- and post-

eccentric exercise. Further paired t-tests were used to determine significant differences in

physiological responses at the V˙

corresponding to the pre-eccentric exercise GET.

Pearson’s product moment correlations were used to examine the relationship between

GET and Tlac values. All data were analysed using the statistical software package SPSS

for Windows (version 13) with statistical significance set at 0.05.

100

5.4 Results

Indicators of muscle damage

There were significant changes in all indicators of muscle damage following eccentric

exercise. Table 5.1 shows changes in muscle soreness, CK activity and isokinetic peak

torque before and after eccentric exercise.

Table 5.1

Changes in indicators of muscle damage. Mean ± SD values before (pre) and

at 30 min, 24 h and 48 h after eccentric exercise.

Measured variable pre 30 min 24 h 48 h

Soreness (1-10 VAS) 0.4 ± 0.4 4.4 ± 2.2* 5.8 ± 2.0* 6.4 ± 3.0*

CK activity (U/L) 180 ± 61 224 ± 103 637 ± 413* 350 ± 151*

Peak Torque (N.m) 30 deg.s-1 300 ± 53 240 ± 68* 248 ± 62* 254 ± 72*

* significantly different from pre-eccentric exercise value (P < 0.05).

Muscle soreness increased 30 min after eccentric exercise with the highest values reported

at 48 h (F (3, 27) = 21.01 P < 0.001). CK activity increased after eccentric exercise, with the

highest values observed at 24 h (FGG (1.38,12.39) = 9.61 P < 0.05). Isokinetic peak torque (30

deg.s-1) decreased by 20% at 30 min and was still 15% lower than baseline values at 48 h

(F (3, 27) = 17.96 P < 0.001).

Ramp Incremental Exercise

Peak values attained during the ramp tests pre- and post- eccentric exercise are shown in

Table 5.2. Time to exhaustion and associated WRpeak values were decreased following

eccentric exercise (t(9) = 2.62, P < 0 .05). There were no significant changes in V˙ E peak,

101

V˙ O 2

peak,

V˙ CO2 peak, HRpeak, RERpeak, [La]peak or RPEpeak (all P > 0.05). Table 5.3 shows

changes in variables associated with GET before and 48h after eccentric exercise. GET

occurred earlier and thus at a lower WR (t(9) = 3.74, P = 0.005) and V˙ O2 (t(9) = 2.57, P =

0.030). V˙ CO2 values at GET were also significantly lower (t(9) = 2.54, P = 0.032).

Table 5.2 Peak values attained during ramp exercise tests. Mean ± SD values before

(pre) and 48 h after (post) eccentric exercise

pre post

Time to exhaustion (s) 682 ± 71 635 ± 86*

WRPeak (W) 341 ± 36 318 ± 43*

peak (l.min-1)

peak

(l.min

-1

)

peak

(l.min

-1

)

HRpeak (beats.min-1) 182 ± 12 179 ± 13

RERpeak 1.35 ± 0.10 1.33 ± 0.12

[La]peak (mmol.l-1) 6.05 ± 1.36 5.64 ± 1.72

RPEpeak (Borg, 6-20 scale) 19.4 ± 0.5 19.1 ± 1.0

Peak minute ventilation ( V˙ E peak), V˙ O2 peak, V˙ CO 2 peak HRpeak and RERpeak were the highest 30s

average values attained during the ramp exercise tests.

* significantly different from pre-eccentric exercise value (P < 0.05).

145 ± 22 140 ± 35

3.34 ± 0.38 3.27 ± 0.35

4.47 ± 0.45 4.33 ± 0.56

102

Table 5.3 Values attained at the gas exchange threshold (GET) during ramp exercise

tests. Mean ± SD values before (pre) and 48 h after (post) eccentric exercise

pre post

Time (s) 262 ± 55 200 ± 37*

WR (W) 136 ± 27 105 ± 19*

(l.min-1)34.2 ± 6.1 31.7 ± 4.6

-1

O2 (l.min

)1.58 ± 0.26 1.41 ± 0.14*

CO2

(l.min-1)1.35 ± 0.24 1.20 ± 0.18*

[La] (mmol.l-1) 1.71 ± 0.38 1.55 ± 0.21

RPE (Borg 6-20 scale) 10.5 ± 0.9 10.6 ± 1.4

* significantly different from pre-eccentric exercise value (P < 0.05).

Figure 5.2 shows the V˙ CO2 versus V˙ O2 (panel A) and blood [lactate] (panel B) responses

of a representative participant during ramp incremental exercise before and after eccentric

exercise; note GET is shifted to a substantially lower V˙ O2 after eccentric exercise whereas

Tlac is essentially unchanged. There were no significant differences in V˙ E , [La] or RPE

at GET before and 48h after eccentric exercise (P > 0.05).

103

Figure 5.2 Representative response of V˙ CO2

V˙

(panel A) and blood [lactate]

(panel B) showing the region of interest, pre- (●) and 48 h post- (○) eccentric exercise

respectively. Best-fit S1 slopes and vertical arrows indicating the gas exchange threshold

(GET, panel A) and lactate threshold (Tlac, panel B) illustrate changes in the GET but not

the Tlac response, pre- (solid line) and post- (dashed line) eccentric exercise.

104

Figure 5.3 shows RPE responses before and after eccentric exercise. The Tlac occurred at a

similar work rate (pre, 161 ± 19 W; post, 158 ± 22 W, P> 0.05) and V˙ O2 (pre, 1.90 ± 0.20

l.min-1; post, 1.88 ± 0.15 l.min-1, P > 0.05) after eccentric exercise. The V˙ Oat GET was

significantly correlated with V˙ O2 at Tlac before (r = 0.78, P < 0.05) but not after eccentric

exercise (r = 0.50, P > 0.05).

There was no significant correlation between changes in

GET and Tlac values pre and post eccentric exercise (r = 0.53, P > 0.05).

Figure 5.3 Changes in ratings of perceived exertion (RPE) as a function of V˙ O2 , pre-

(●) and 48 h post- (○) eccentric exercise respectively. The vertical arrow indicates the gas

exchange threshold (GET) pre-eccentric exercise. There is a 7% increase (P < 0.05) in RPE

48 h post eccentric exercise at the V˙ O2 value of the pre-eccentric exercise GET.

* Significantly different from pre-eccentric exercise value (P < 0.05)

105

However, RPE increased significantly at the

V˙ O2

corresponding to the pre-eccentric

exercise GET. V˙ O2 was unchanged by the damage protocol and as anticipated, increased

as a linear function of work rate before and 48 h after eccentric exercise. A comparison of

responses before and 48 h after eccentric exercise at the V˙ O2 value at which pre-eccentric

exercise GET occurred revealed several significant differences (Figure 5.4).

Specifically,

there were increases in V˙ E

(t(9) = -2.88, P < 0 .05), V˙ CO2 (t(9) = -3.31, P < 0 .05), RER (t(9)

= -2.92, P < 0 .05), V˙ E / V˙ O2

(t(9) = -2.63, P < 0 .05), fR (t(9) = -2.49, P < 0 .05), and RPE

(t(9) = -2.40, P < 0 .05). There were no differences in V˙ E / V˙ CO2 , VT or [La] (P > 0.05).

Figure 5.4 Percentage changes in minute ventilation ( V˙ E ), expired CO2 ( V˙ CO2 ),

respiratory exchange ratio (RER), ventilatory equivalent for O2 ( V˙ E / V˙ O2

),ventilatory equivalent for CO2 ( V˙ E / V˙ CO2 ), breathing frequency (fR) and tidal

volume (VT) at the V˙ O2 value of the pre-eccentric exercise GET. Values are mean (±

SD).

* Significantly different from pre-eccentric exercise value (P < 0.05)

106

5.5 Discussion

The principal original finding of this investigation is that eccentric, muscle-damaging

exercise dissociates the V˙ E

and gas exchange responses to ramp incremental exercise from

the blood lactate response. While we believe these findings to be novel with respect to exercise-

induced muscle damage (EIMD), previous research has demonstrated that the GET and

lactate threshold (Tlac) may be dissociated by certain experimental protocols including

exercise training (Poole & Gaesser, 1985) and glycogen depletion (Hughes et al., 1982).

Thus our findings provide evidence to suggest that following eccentric exercise the control of

pulmonary ventilation is influenced by altered or additional stimuli, possibly of neurogenic

origin.

As anticipated, the [La] response to ramp incremental cycle exercise was not altered by the EIMD

intervention. This finding supports the contention that elevated blood [La] is not an

inevitable consequence of EIMD and is, therefore, unlikely to be linked causally to the

augmented V˙ E

response observed herein and elsewhere (Twist & Eston, 2009) (Chapter 4).

Instances of increased blood [La] following eccentric exercise have been linked with a putative

switch to more glycolytic energy production possibly as a result of an increased reliance on

type II motor unit recruitment (Braun & Dutto, 2003; Chen et al., 2007b; Gleeson et al.,

1995, 1998). However, it has been demonstrated that EIMD does not compromise skeletal

muscle oxidative function (Walsh et al., 2001) or alter pulmonary

V˙ O2 kinetics (Schneider et al., 2007).

Our findings contrast with those reported in the only previous study to investigate responses to

incremental exercise after eccentric exercise (Gleeson et al., 1998).

Gleeson

107

and co-authors (1998) reported no change in endurance time, V˙ O2 , V˙ E , or RER responses

but found [La] to be higher 48 h after completing a bench-stepping intervention which

provoked only mild soreness. Differences in the damage intervention, the incremental

protocol employed and the level of muscle soreness induced may account for the disparity

in V˙ E and [La] responses between investigations.

There was a small and statistically non-significant difference between the pre-eccentric

exercise work rates for the lactate and gas exchange thresholds, with the gas exchange

threshold occurring slightly earlier. It should be recognised that the dynamics of blood

lactate production and clearance (and hence appearance of lactate in the blood) may differ

from the more immediate stimuli for changes in gas exchange and ventilation during ramp

incremental exercise. Moreover, gas exchange was measured on a continuous breath-by-

breath basis whereas blood samples for [La] determination samples were collected at

discrete 1-min intervals. This may increase the potential for disagreement between the

lactate and gas exchange thresholds.

The cause and effect relationship between Tlac (cause) and GET (effect) is fundamental to

the traditional “anaerobic threshold” hypothesis (Beaver et al., 1986; Wasserman et al.,

1973, 1990).

However, certain experimental conditions have been demonstrated to elicit a

dissociation of [La] and

V˙ E responses. Under conditions of acute hypoxia (Ozcelik &

Kelestimur, 2004) and following prior volitional hyperventilation (Ozcelik et al., 1999)

GET is reduced in comparison to the normal, control condition, without a concomitant

decrease in Tlac.

It has been proposed that a ‘pseudo-threshold’ (Whipp, 1987) is evoked

by a ‘wash-in’ of CO2 to the depleted body stores (hyperventilation) or enhanced carotid

108

body chemosensitivity to CO2 (hypoxia). However, the reduction in GET observed in the

present study does not appear to result from humoral stimuli arising from alterations in

blood [La]. Previous investigations reporting a dissociation of V˙ E and [La] responses to

exercise have suggested that V˙ E

may have been influenced by differences or alterations in

the extent of neurogenic control. Patients suffering from McArdle’s disease, a condition

where

lack

muscle

phosphorylase

precludes

increases

[La]

during

exercise,

demonstrate a dissociation of V˙ E

and [La] responses during incremental cycling (Hagberg

et al., 1982; Paterson et al., 1990). Similarly, under conditions of reduced muscle glycogen

content (Hughes et al., 1982; Sabapathy et al., 2006) and following a period of exercise

training (Poole & Gaesser, 1985)

V˙ E and [La] responses to exercise are dissociated.

However, the existence of a causal link between bicarbonate buffering of lactic acidosis and

increases in V˙ E has recently been supported in a study which used DCA to reduce [La]

during incremental exercise (Wilkerson et al., 2009). While lactic acidosis may provide an

important stimulus to V˙ E

it is only one of many factors that can influence the V˙ E

response

and therefore modulate GET (Dempsey et al., 2006; Ward, 2000; Whipp et al., 1981). It

should be mentioned here that while under normal control conditions it is considered that

increased non-metabolic CO2 production from the bicarbonate buffering of lactic acidosis

stimulates the increased

V˙ E (Wasserman et al., 1973, 1990), in the present study the

relative hyperventilation following eccentric exercise might have increased

V˙ CO2

‘blowing off’ CO2 from the body stores (Ozcelik et al., 1999) thus reducing the GET as

established using the V-slope method (Beaver et al., 1986). Thus we speculate that the

augmented V˙ E

response and reduced GET observed herein likely result from non-humoral

stimuli originating in the damaged, exercising muscle.

109

The eccentric exercise protocol employed in this study is known to be effective in inducing

damage (Byrne & Eston, 2002a, 2002b) (Chapter 4). The primary event which leads to

muscle damage from unaccustomed eccentric exercise involves the disruption of

sarcomeres and myocyte membranes leading to dysfunction of the excitation-contraction

(E-C) mechanism (Proske & Morgan, 2001). As a consequence the immediate and

prolonged reductions in peak torque observed in the present investigation may reflect

damage to the E-C mechanism. Furthermore the significant increase in plasma CK activity

is indicative of increased membrane permeability. While EIMD does not appear to disrupt

intrafusal fibres as demonstrated by the unaltered sensitivity of muscle spindle or Golgi

tendon afferents (Gregory et al., 2002, 2004), the disruption to extrafusal fibres may

provide mechanical stimulation to fine myelinated (group III) and unmyelinated (group IV)

afferents (Avela et al., 1999; Komi, 2000; Taguchi et al., 2005).

These thin-fibre afferents

can exert a significant influence on V˙ E

during dynamic exercise (Matieka & Duffin, 1995)

and may have provided an important additional drive to V˙ E leading to the reduction in

GET observed in the present study. Similarly, structural and functional disruption to local

microvasculature following eccentric exercise (Kano et al., 2005) has been implicated in

augmenting the V˙ E response via stimulation of group III and IV afferents (Haouzi et al.,

2004). We have previously used an identical eccentric exercise protocol to that employed

herein to induce damage and have observed disruption in the matching of O2 delivery (

Q˙ O2 ) to O2 utilisation ( V˙ O2 ), consistent with such microvascular dysfunction,

conjunction with an augmented V˙ E response (Chapter 4) .

110

Muscular pain is understood to provide an important stimulus to the ventilatory response

via the mechanical stimulation of nociceptive muscle afferents (Duranti et al., 1991).

Excitation of these afferents via painful electrical muscular stimulation or ischemic muscle

pain elicits a reflex increase in breathing frequency (fR) and minute ventilation (Duranti et

al., 1991) whilst concurrently depressing the central neural drive to ventilation (Waldrop et

al., 1982). As the mechanical sensitivity of group III and IV thin fibre muscle afferents is

augmented by experimental muscle pain as detailed above, so it may be increased by the

mechanical hyperalgesia, the sensations of muscle tenderness and movement-induced pain,

that result from unaccustomed eccentric exercise.

Thus the soreness experienced by our

participants during muscle activation may have contributed to the augmented fR and V˙ E

observed following eccentric exercise via afferent neural reflexes.

The elevated ratings of perceived exertion (RPE) reported by our participants corroborate

previously reported findings during constant load cycle exercise after eccentric exercise

(Twist & Eston, 2009) (Chapter 4) and may account for the reduction in time to exhaustion.

This decrease and the associated reduction in WRpeak values are consistent with

observations of impaired endurance performance brought about by the effects of eccentric

exercise (Marcora & Bosio, 2007; Twist & Eston, 2009) (Chapter 4). Submaximal

differences in V˙ E , V˙ CO2 , RER and RPE during ramp exercise are resolved at maximal

exercise albeit at a lower work rate and exercise time. This observation supports the

proposition that the rating of perceived exertion scales with exercise duration (Crewe et al.,

2008; Eston et al., 2007; Faulkner et al., 2008; Joseph et al., 2008) and also that a strong

link exists between V˙ E and RPE responses to cycling following eccentric exercise (Chapter

4).

111

5.6 Conclusion

In conclusion, a prior bout of muscle damaging eccentric exercise augments the V˙ E

response to ramp incremental cycling leading to a reduction in GET in the absence of

altered blood [La]. The resultant dissociation between GET and Tlac indicates that the two

phenomena are not causally linked in the presence of EIMD.

We propose that the increase

in V˙ E

and reduction in GET 48 hours after muscle-damaging eccentric exercise are evoked

most likely by increased activation of group III and IV afferents stimulated via mechanical

disruption of muscle fibres and local microvasculature and are not the result of altered

blood [La].

The contents of this chapter are currently under review for publication:

Davies RC, Eston RG, Fulford J, Rowlands AV and Jones AM. Muscle damage alters

the metabolic response to dynamic exercise in humans: a 31P-MRS study. Currently

under review.

112

CHAPTER 6

THE 31P-MRS METABOLIC RESPONSE TO INCREMENTAL

EXERCISE FOLLOWING ECCENTRIC,

MUSCLE-DAMAGING EXERCISE

113

6.1 Abstract

This study used 31P-magnetic resonance spectroscopy (31P-MRS) in an attempt to reveal

any link between changes in muscle metabolism and the limitations to exercise tolerance in

humans with and without exercise-induced muscle damage (EIMD). Ten healthy,

physically active men performed incremental knee extensor exercise inside the bore of a

whole-body MRS before (pre) and 48 h after (post) performing 100 squats with a load

corresponding to 70% of body mass. Time to exhaustion was significantly reduced

following EIMD (519 ± 56 and 459 ± 63 s, pre and post EIMD, respectively). End exercise

pH (pre: 6.75 ± 0.04 post: 6.83 ± 0.04) and [PCr] (pre: 7.2 ±1.7 post: 14.5 ± 2.1 mM)

values were higher following EIMD (both P <.05). However, end exercise [Pi] was not

significantly different following EIMD (pre: 19.7 ± 1.9 post: 21.1 ± 2.6 mM, P > .05).

Resting [Pi] values (pre: 4.7 ± 0.8 post: 6.7 ± 1.7 mM) and consequently [Pi]:[PCr] values

(pre: 0.12 ± 0.02 post: 0.18 ± 0.05) were significantly elevated following EIMD and these

mean differences were maintained during incremental exercise (all P <.05). In contrast,

[PCr] and pH values were not different pre and post EIMD at rest or during incremental

exercise (all P >.05). Findings indicate that alterations in phosphate metabolism,

specifically increases in resting [Pi] that are maintained during exercise, may contribute to

the reduced exercise tolerance experienced with EIMD.

114

6.2 Introduction

Unaccustomed eccentrically-biased exercise results in substantial alterations to skeletal

muscle structure and function. Morphological changes include disruption of the

cytoskeleton, sarcolemma and T-tubules (Fridén and Lieber, 2001), and changes to

capillary geometry (Kano et al., 2004). The loss of sarcolemmal integrity results in an

efflux of intramyocyte proteins into the bloodstream (Hortobágyi & Denahan, 1989) and an

influx of extracellular Ca2+ into the sarcoplasm (Armstrong, 1984). These changes are

associated with an acute inflammatory response and delayed onset muscle soreness

(DOMS) (MacIntyre et al., 1996) and are collectively referred to as exercise-induced

muscle damage (EIMD).

Performance decrements associated with EIMD include a reduction in maximal force-

generating capacity (Clarkson et al., 1992) and a shorter time to exhaustion (Asp et al.,

1998; Carmichael et al., 2005, 2006). Carmichael et al. (2005, 2006) have reported

decreases in treadmill run time to fatigue in mice following a bout of downhill running.

The decline in performance was associated with increases in the inflammatory cytokine,

interleukin 1 β (IL-1β) within regions of the brain responsible for movement, motivation,

perception of effort and pain (Carmichael et al., 2005). Central fatigue factors such as the

enhanced production of inflammatory cytokines have also been implicated in reduced time

trial running (Marcora and Bosio, 2007) and cycling performances in humans (Twist and

Eston, 2009,) where an elevated perception of exertion appeared to mediate performance

following eccentric exercise. Peripheral fatigue factors originating within the damaged

muscle tissue may also be involved in the reduced time to exhaustion following eccentric,

muscle-damaging exercise.

Using muscle biopsy procedures, Asp et al. (1998) reported

115

decreases in muscle glycogen content in human subjects following muscle-damaging

exercise which were associated with a 23% reduction in maximal work capacity during

incremental knee extensor exercise. Other observed changes in metabolic function

following eccentric exercise include impaired muscle glycogen resynthesis (Asp et al.,

1995, 1998) and an elevated blood lactate response during exercise (Gleeson et al., 1995,

1998; Asp et al., 1998). It is feasible that these changes reflect a shift in the muscle

metabolic profile to an increased reliance on non-oxidative metabolism, contributing to the

decreased endurance capacity following EIMD (Asp et al., 1998). However, the precise

nature of the accelerated fatigue development experienced with EIMD is poorly

understood.

Non-invasive evaluation of muscle metabolism can be achieved using 31P-magnetic

resonance spectroscopy (31P-MRS). Several studies have used this technology to

demonstrate an increase in the resting inorganic phosphate (Pi) to phosphocreatine (PCr)

ratio following EIMD, suggestive of an increase in resting muscle metabolism (McCully

1992, Rodenburg et al., 1995; Lund et al., 1998a, 1998b). Several of the candidate

mechanisms for the accelerated development of fatigue and reductions in peak power

observed following eccentric exercise, including increases in Pi and ADP and decreases in

pH, can be measured using 31P-MRS. Rodenburg et al. (1995) reported no difference in the

Pi:PCr ratio, pH or peak power during graded knee extensor exercise performed 24 h after a

bout of stepping exercise designed to induce EIMD. However, the authors concluded that

the lack of change in several markers of muscle damage indicated that the muscle-

damaging protocol employed was not sufficiently severe to alter muscle metabolism

(Rodenburg et al., 1995).

116

It has been demonstrated that an effective muscle-damaging protocol reduces time to

exhaustion during high-intensity cycling (Chapter 4). However the mechanisms underlying

the reduced endurance capacity following muscle-damaging exercise remain to be

determined. Central mechanisms which may potentially influence the increased

development of fatigue, such as increased inflammatory cytokine production are difficult to

evaluate during dynamic exercise in the human model. Putative peripheral fatigue

mechanisms including an increased rate of [PCr] depletion, an increased accumulation of

[Pi] and [ADP] and a decreased rate of fall in pH can all be assessed using 31P-MRS.

Following a period of endurance training, incremental knee extensor exercise tolerance is

enhanced with the relationships of [Pi]:[PCr], [PCr] and pH against time demonstrating a

rightward shift (Jones et al., 2007). It is possible that following a bout of muscle-damaging

exercise the reduced exercise tolerance observed could be associated with a leftward shift

in these 31P metabolite-time profiles. Alternatively, an unchanged muscle metabolite

response would indicate that other, potentially central mechanisms may be responsible for

any decrements in endurance capacity. The purpose of this study was therefore to

investigate the effect of a well-defined bout of eccentric, muscle-damaging exercise

(Chapter 3) on changes in muscle metabolism during dynamic incremental knee extensor

exercise. We used 31P-MRS to test the hypothesis that exercise-induced muscle damage

(EIMD) alters the muscle metabolic response to dynamic exercise and thus limits exercise

tolerance in humans.

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6.3 Methods

Subjects

Ten healthy, physically active male subjects (age, 22 ± 4 years; mass, 78.2 ± 8.8 kg; height,

1.79 ± 0.08 m) volunteered to participate in this study. All participants were asymptomatic

of illness and pre-existing injuries and had not performed any resistance training of the

lower limbs within the previous six months. Participants provided written informed

consent to participate in the study which was approved by the Institutional Ethics

Committee (See appendices B, C and D for exemplar participant information sheet,

participant consent form and ethical approval certificate).

Assessment of muscle damage

All indicators of muscle damage, perceived muscle soreness (using a 0-10 visual analogue

scale (VAS)), creatine kinase (CK) activity and isokinetic peak torque (30 deg.s-1), were

measured in the order listed, immediately before, and 24 and 48 h after performing the

eccentric, muscle-damaging exercise protocol. For further details of these procedures

please refer to Chapter 3.

Experimental procedures

Following the assessment of muscle damage, single -legged, knee-extension exercise tests

were completed at the same time of day ± 1 h for each participant, before and 48 h after

performing eccentric, muscle-damaging exercise. The dynamic knee-extensor exercise

tests were conducted in the prone position with the subjects positioned inside a whole body

MRI system. A 6-cm 31P transmit-receive surface coil was placed within the subject bed,

and the subject was asked to lie on it such that the coil was centred over the quadriceps

118

muscle of the leg to be exercised. Subjects were then secured to the ergometer bed with

Velcro straps at the thigh, buttocks, and lower back to minimise extraneous movement

during the protocol. The foot of the leg to be exercised was connected to a pulley system

that permitted a nonmagnetic weight to be lifted and lowered and work rate to be calculated

(Figure 6.1).

Exercise was performed at a rate of 40 repetitions/min with the subjects lifting and

lowering the mass over a distance of ~0.22 m in accordance with a visual cue projected

onto the front wall of the scanner room. The contraction phase of the knee extensors and

the 31P-MRS interrogation of the quadriceps occurred in unison. After a 2-min period of

rest, the subjects commenced knee-extension exercise against an initial basket load of 1 kg.

Thereafter, the basket load was increased by 0.5 kg every 30 s until the subjects were no

longer able to maintain the kicking frequency at 40 repetitions/min. The subjects received

strong verbal encouragement to continue for as long as possible while maintaining

appropriate form.

Eccentric, muscle-damaging exercise protocol

Participants completed 100 (Smith) squats, performed as 10 sets of 10 repetitions with the

load on the bar corresponding to ~70% of each participant’s body mass. Prior to

commencing, all participants were instructed in correct and safe lifting technique. The bar

was positioned on the participant’s shoulders and feet were positioned under the bar, with

the back straight and legs fully extended (knee = 180°). The descent phase involved

eccentric action of the knee extensors to lower the bar to a knee angle of just past 90°. The

Pulley system

Load basket

Subject lying in a prone

position within the core of

the whole body scanner.

The right foot is attached

to the pulley system.

119

lifting phase involved concentric action to return the bar to the starting position. For further

details of these procedures please refer to Chapter 3.

Figure 6.1 The MRS knee extensor ergometer showing pulley system and load basket.

Measurements

MRS measurements

MRS was performed in the Peninsula Magnetic Resonance Research Centre using a 1.5-T

superconducting magnetic resonance scanner (Philips Gyroscan Clinical Intera, Philips

Medical Systems, Best, Netherlands). Initially, fast-field echo images were acquired to

120

determine whether the muscle was positioned correctly relative to the coil. This was aided

by placing cod liver oil capsules, which yield high-intensity signal points within the image,

adjacent to the coil, allowing its orientation relative to the muscle volume under

examination to be assessed. A number of preacquisition steps were carried out to optimise

the signal from the muscle under investigation. Tuning and matching of the coil were then

performed, followed by an automatic shimming protocol undertaken within a volume that

defined the quadriceps muscle. The muscle volume from which signal originates is dictated

by the sensitive volume of the coil itself. This approximates to the physical size of the coil

and thus corresponds to a cylinder of 6cm diameter and 6cm deep adjacent to the coil.

Outside of this volume, muscle will still contribute to the signal but to an extent that drops

off rapidly as you move away from the sensitive region. To ensure that the examined

muscle was consistently at the same point relative to the coil during exercise, the subject

was visually queued via a display consisting of two vertical bars, one that moved at a

constant rate with a frequency of 0.67 Hz and one that monitored foot movements via a

sensor present within the pulley to which they were connected. Thus the subject

endeavoured to match the movements of these two bars. The work done by the subjects

was recorded via a nonmagnetic strain gauge present within the pulley mechanism. Before

exercise, during exercise, and during recovery, data were acquired every 1.5 s, with a

spectral width of 1,500 Hz and 1,000 data points. Phase cycling with eight phase cycles

was employed, leading to a spectra being acquired every 12 s. The subsequent spectra were

quantified via peak fitting, assuming prior knowledge, using the jMRUI (version 2)

software package and the AMARES fitting algorithm (Vanhamme et al., 1997; Naressi et

al., 2001).

121

Spectra were fitted assuming the presence of the following peaks: Pi, phosphodiester,

phosphocreatine (PCr), α-ATP (2 peaks, amplitude ratio 1:1), γ-ATP (2 peaks, amplitude

ratio 1:1), and β-ATP (3 peaks, amplitude ratio 1:2:1). In all cases, relative amplitudes were

corrected for partial saturation because of the short repetition time relative to the

longitudinal relaxation time constant T1. Absolute concentrations were determined by

calculating the size of peak areas relative to β-ATP which was set at 8.2 mM. The ratio of

Pi to PCr was determined from the respective Pi and PCr spectral areas as obtained during

the quantification procedure. Intracellular pH was calculated from the chemical shift of the

Pi spectral peak relative to the PCr (Moon and Richards, 1973). ADP concentrations were

calculated as described by Kemp et al., (2001) taking into account the pH dependency of

the binding of H+, K+ and Mg2+. To determine the intracellular threshold (IT) during

incremental exercise, piecewise linear regression was used. Briefly, different two-line

combinations were fitted to the [Pi]/[PCr]-work rate and pH-work rate relationships until

the lowest sum of squared residuals was found (Hogan et al., 1983; Marsh et al., 1991). The

point at which this particular two-line combination intersected was accepted as the IT.

Statistical analysis

Changes in the indicators of muscle damage (perceived muscle soreness, creatine kinase

activity and isokinetic peak torque) were analysed using a series of one-way repeated

measures ANOVAs. As the CK activity data were not normally distributed these values

were log-transformed prior to statistical analysis (Twist & Eston, 2005) (see appendix H).

Paired t-tests were used to determine significant differences in end exercise values

between the two conditions. Where data were not normally distributed Wilcoxon tests were

also run. Changes in the MRS measurements (Pi, PCr, Pi:PCr ratio and pH) at rest and

122

after 2, 4 and 6 min of incremental exercise were examined using separate two-way fully

repeated measure ANOVAs (condition x time). The assumption of sphericity was

evaluated using Mauchly’s test. Where sphericity was violated (P < 0.05), the Greenhouse-

Geisser (GG) correction factor was applied. All data were analysed using the statistical

software package SPSS for Windows (version 13) with statistical significance set at 0.05.

Values are means ± SD.

6.4 Results

Muscle damage

There were significant changes in all indicators of muscle damage following eccentric

exercise. Table 6.1 shows changes in muscle soreness, CK activity and isokinetic peak

torque before and after eccentric exercise. Muscle soreness increased 24 h after eccentric

exercise with the highest values reported at 48 h (F (2, 18) = 26.22, P < 0.05). Plasma CK

activity increased after eccentric exercise, with the highest values observed at 24 h (FGG (1.1,

9.9) = 15.02, P < 0.05).

Isokinetic peak torque (30 deg.s-1) decreased by 15% at 24 h and

remained 11% lower than baseline values at 48 h (F (2, 18) = 14.33, P < 0.05).

123

Table 6.1 Changes in indicators of muscle damage. Mean ± SD values and (range)

before and 24 h and 48 h after eccentric exercise.

Measured Variable before 24 h 48 h

Soreness (0-10 VAS)

0.5 ±

0.3

(0.1 –

1.1)

5.0 ±

2.0*

(1.8 –

8.2)

5.5 ± 3.2*

(1.6 – 9.7)

CK activity (U/L) 178 ±

(97 – 270)

798 ±

692*

(196 –

2481)

409 ± 264*

(213 – 1088)

Peak Torque (N.m)

30 deg.s

-1

310 ± 47

(227 –

366)

265 ±

66*

(113 –

340)

275 ± 77*

(115 - 361)

Soreness, visual analogue scale (VAS) 0–10. CK, creatine kinase.

* Significantly different from pre-eccentric exercise value (P < 0.05).

MRS measurements

As anticipated, time to exhaustion (519 ± 56 and 459 ± 63 s, pre and post muscle damage

respectively) and associated peak work rate values (29 ± 4 and 25 ± 4 W, pre and post

muscle damage respectively) attained during the incremental knee extensor exercise were

significantly reduced following muscle damage (t(9) = 4.85, P < 0.05 and t(9) = 5.21, P <

0.05, respectively). End exercise values are presented in Table 6.2. The data for end

exercise [Pi]:[PCr] and ADP were not normally distributed therefore Wilcoxon tests were

used to analyse these data. However results did not differ from t-test results therefore for

consistency the results of the paired t-tests are presented.

124

Table 6.2 Muscle metabolic responses at rest and during incremental exercise before

(Pre) and 48h after (Post) eccentric, muscle-damaging exercise. Values are means ± SD.

Measured

Variable Resting 2 min 4 min 6 min End

[PCr] (mM) †Pre

38.3 ± 1.2 35.0 ± 2.1 30.7 ± 3.1 24.3 ± 4.0 7.2 ± 1.7

Post

37.9 ± 1.4 34.1 ± 2.6 31.3 ± 3.0 25.5 ± 4.8 14.5 ± 2.1*

[Pi] (mM) †Pre

4.7 ± 0.8 5.6 ± 1.1 8.0 ±1.9 10.6 ± 3.0 19.7 ± 1.9

Post ‡

6.7 ± 1.7 7.5 ± 2.1 10.4 ± 4.0 14.8 ± 6.5 21.1 ± 2.6

[Pi]:[PCr] ratio†Pre

0.12 ±

0.02

0.16 ±

0.03

0.26 ±

0.05

0.45 ±

0.13

2.09 (1.74-14.4) ††

Post ‡

0.18 ±

0.05

0.22 ±

0.07

0.34 ±

0.15

0.62 ±

0.34

1.46 (0.85-2.29) ††*

†

Pre 7.02 ±

0.03

7.06 ±

0.04

7.03 ±

0.04

6.96 ±

0.06

6.75 ± 0.04

Post

7.02 ±

0.03

7.07 ±

0.03

7.04 ±

0.05

6.99 ±

0.09

6.83 ± 0.04*

† Significant main effect for time (resting, 2, 4 and 6 min) (P < 0.05)

‡ Significant main effect for condition (Pre and Post) (P < 0.05)

* Significantly different from Pre-eccentric exercise (P < 0.05)

†† End exercise [Pi]:[PCr] ratio data were not normally distributed therefore values are

presented as median (interquartile range).

Table 6.3 Intracellular Threshold (IT) values before (Pre) and 48h after (Post) eccentric,

muscle-damaging exercise. Values are means ± SD.

Measured Variable Pre Post

Time @ IT (s)

326 ± 77 316 ± 65

[Pi]:[PCr] ratio @ IT

0.30 ± 0.14 0.31 ± 0.10

pH @ IT

7.07 ± 0.02 7.08 ± 0.02

125

End exercise pH and [PCr] values were higher (t(9) = -2.34, P < 0.05 and t(9) = -4.49, P <

0.05, respectively) and end exercise [Pi]:[PCr] values were lower (t(9) = 2.346, P < 0.05,)

following eccentric exercise. However, end exercise [Pi] and ADP were not significantly

different following eccentric exercise (t(9) = -0.496, P > 0.05 and t(9) = 1.82, P > 0.05,

respectively). The IT was not significantly altered by eccentric exercise (t(9) = 0.51, P >

0.05). The time, [Pi]:[PCr] and pH values when the IT occurred are presented in Table 6.3.

Prior to eccentric exercise, all ten participants completed a minimum of 7 min exhaustive

incremental exercise. However, 48 h after eccentric exercise the minimum time achieved

by all participants was reduced to 6 min. Thus, the first 6 min of incremental exercise were

analysed in order to examine changes in muscle metabolic responses during exercise.

0 60 120 180 240 300 360 420 480 540 600

Time (s)

Figure 6.2 [PCr] response of a representative participant, pre- and post-eccentric,

muscle damaging exercise. Vertical arrows indicate time to exhaustion in the two exercise

conditions. The solid line represents time to exhaustion pre-eccentric exercise; the dashed

line represents time to exhaustion post-eccentric exercise.

Pre-eccentric exercise

Post-eccentric

exercise

[PCr] (mM)

126

Muscle metabolic responses at rest and during incremental exercise are presented in Table

6.2 (see appendix I for graphical representation of these data). There was a significant

main effect for time for all muscle metabolic responses, with [PCr] and pH values

declining, and [Pi] and [Pi]:[PCr] values increasing as the incremental exercise progressed

(all P < 0.05). There was a significant main effect for condition for [Pi] (F(1,9) = 7.080, P <

0.05). Resting [Pi] values were significantly elevated and this mean difference was

maintained during the first 6 min of incremental exercise.

2.5

1.5

0.5

0 60 120

180

240

300

360

420

480

540

600

Time (s)

Figure 6.3 [Pi]:[PCr] response of a representative participant, pre- and post-eccentric,

muscle damaging exercise. Vertical arrows indicate time to exhaustion in the two exercise

conditions. The solid line represents time to exhaustion pre-eccentric exercise; the dashed

line represents time to exhaustion post-eccentric exercise.

Pre-eccentric exercise

Post-eccentric

exercise

[Pi]:[PCr]

127

As a direct consequence, [Pi]:[PCr] values were also elevated at rest and during the first 6

min of exercise (F(1,9) = 5.908, P < 0.05). In contrast, [PCr] and pH values were not

different between conditions at rest and during incremental exercise (both P > 0.05). There

were no significant interactions of condition and time for any muscle metabolic responses.

Changes in [PCr], [Pi]:[PCr] and pH are illustrated for a typical participant in Figures 6.2 -

6.4.

7.15

7.1

7.05

6.95

6.9

6.85

0 100 200 300 400 500 600

Time (s)

Figure 6.4

pH response of a representative participant, pre- and post-eccentric, muscle

damaging exercise. Vertical arrows indicate time to exhaustion in the two exercise

conditions. The solid line represents time to exhaustion pre-eccentric exercise; the dashed

line represents time to exhaustion post-eccentric exercise.

Pre-eccentric exercise

Post-eccentric

exercise

128

6.5 Discussion

To our knowledge, this is the first study to investigate changes in muscle metabolism with

31P-MRS during incremental knee extensor exercise following an effective muscle-

damaging protocol. The principal original finding of this investigation was that the

reduction in time-to-exhaustion consequent to a bout of eccentric, muscle-damaging

exercise was not associated with an accelerated depletion of [PCr] or faster fall in pH.

Specifically, time-to-exhaustion was reduced by 12% following the muscle-damaging

exercise but the temporal changes in [PCr] and pH were similar in the two experimental

conditions such that end-exercise [PCr] and pH were significantly higher 48 h after the

muscle-damaging exercise (Figures 6.1 and 6.3). In contrast, the end-exercise [Pi] was not

significantly different between the pre- and post-EIMD conditions. These results indicate

that the reduced exercise tolerance following EIMD may be related either to the increased

[Pi] that was observed at rest and throughout incremental exercise, or to other unmeasured

peripheral or central factors. Importantly, however, the results allow us to discount a greater

rate of non-oxidative energy metabolism (as inferred from the changes in [PCr] and pH) as

important mediators of the reduced exercise tolerance following EIMD.

Our findings provide novel insights into changes in 31P metabolite responses to dynamic

exercise subsequent to muscle-damaging exercise. Previous research has reported

unaltered Pi:PCr ratio, pH and peak power during incremental exercise following a prior

bout of eccentric exercise (Rodenburg et al. 1995). However, these authors speculated that

the bench-stepping protocol employed to induce damage was not sufficiently strenuous to

elicit changes in exercise metabolism. Indeed, the lack of change in several markers of

muscle damage including maximum power output suggests that their eccentric exercise

129

protocol was ineffective (Rodenburg et al. 1995). Participants in the present study

completed a bout of eccentric, muscle-damaging exercise, comprising 100 squats with the

load on the bar set to 70% body mass (see chapter 3). This eccentric exercise protocol is

known to be effective in inducing damage (Byrne and Eston 2002a, 2002b). Indeed, in the

present study, there were changes in all measured symptoms of muscle damage. The

disruption of sarcomeres and myocyte membranes during unaccustomed eccentric exercise

leads to immediate and prolonged reductions in peak torque due to dysfunction of the

excitation-contraction (E-C) mechanism (Proske and Morgan 2001). The 11% reduction in

isokinetic peak torque observed at 48 h is consistent with previous studies which have

employed this eccentric exercise protocol in reflecting damage to the E-C mechanism

(Byrne and Eston 2002a, 2002b). Furthermore the significant increase in plasma CK

activity is indicative of increased membrane permeability. Impaired maximal force

production may, in part, account for the reduction in time to exhaustion and the associated

14% reduction in peak work rate values. The accelerated rate of fatigue experienced by our

participants is consistent with observations of impaired endurance performance brought

about by the effects of eccentric exercise (Asp et al. 1998; Marcora and Bosio 2007; Twist

and Eston 2009).

Peripheral fatigue factors originating from within the damaged muscle tissue have been

implicated in the reduction in endurance capacity associated with exercise-induced muscle

damage. Specifically, reports of increased blood lactate (Asp et al., 1998; Braun & Dutto,

2003; Chen et al., 2007) and increased utilisation of glycogen stores (Asp et al., 1998)

following eccentric exercise have been attributed to a putative shift towards an increased

reliance on anaerobic energy production leading to impaired endurance performance (Asp

130

et al., 1998; Braun & Dutto, 2003; Chen et al., 2007). However, in the present study, the

PCr response for a given work rate was not significantly altered, although the performance

of incremental knee extensor exercise was impaired.

It is recognised that muscle PCr and

V˙ O2 demonstrate similar kinetic profiles during transitions to higher exercise intensities

(Barstow et al., 1994; Mahler, 1985; Marsh et al., 1993), indicating that the rate of

oxidative phosphorylation is closely linked to PCr hydrolysis (Mahler, 1985, Meyer, 1988).

Thus our observation of an unchanged PCr profile during incremental exercise is consistent

with reports of unchanged

V˙ O2

kinetics following eccentric exercise (Schneider et al.,

2007). Collectively these data indicate that exercise-induced muscle damage does not

compromise oxidative function during dynamic exercise. The reduction in time to fatigue

resulted in significant alterations in end exercise [PCr] values. Prior to the bout of muscle-

damaging exercise the mean depletion of the PCr pool was over 80% at the end of the

incremental exercise, with several subjects almost completely depleting their muscle PCr.

However 48 h after the muscle-damage was induced, participants reached exhaustion

sooner with a mean depletion of the PCr pool of approximately 62%. Thus end-exercise

[PCr] was higher when incremental exercise was performed in the muscle-damaged

condition.

Similarly, intracellular pH demonstrated an unchanged rate of fall during incremental

exercise after muscle-damage was induced. There was no significant difference in the pH

values for a given work rate at 48 h although end exercise pH was higher due to the shorter

time to exhaustion. Furthermore, the unchanged [PCr] and pH responses to incremental

exercise resulted in there being no significant difference in the IT values following

eccentric exercise. Low [PCr] and pH have been implicated in the fatigue process

131

(Westerblad and Allen 2003; Wilson et al. 1988). However, on the evidence of our

observations these effects do not appear to be associated with the reduction in exercise

tolerance that accompanies exercise-induced muscle damage.

At rest, increases in [Pi], resulted in a 50% increase in the resting [Pi]:[PCr] ratio 48 h after

eccentric exercise. These findings are consistent with those of several previous studies

using 31P-MRS in reporting increases in resting [Pi]:[PCr] following eccentric exercise

(McCully 1992, Rodenburg et al., 1995; Lund et al., 1998a, 1998b). Elevated resting [Pi]

such as observed herein and elsewhere (McCully 1992, Rodenburg et al., 1995; Lund et al.,

1998a, 1998b) may be interpreted as an increase in muscle metabolism. The repair and

remodelling of tissue damaged via eccentric exercise such as that reported by Yu et al.

(2002) and Yu and Thornell (2002) could lead to an increase in resting muscle metabolism.

Furthermore, depleted resting muscle glycogen content following eccentric exercise,

particularly the content of the preferentially damaged type II fibres, has been attributed to

increased resting muscle glycogen utilisation (Asp et al., 1998). An alternative explanation

for the increase in [Pi]:[PCr] which, importantly, results from increased [Pi] but not

decreased [PCr], may be linked to the breakdown of muscle tissue consequent to eccentric

exercise. Disturbances in intracellular Ca2+ following eccentric exercise have been reported

with concomitant activation of calcium-activated proteolytic pathways (Belcastro, 1993;

Belcastro et al. 1998). Thus the observed increase in resting [Pi] may not be due to

increases in muscle metabolism but to the degradation of muscle proteins.

Increases in resting [Pi] and [Pi]:[PCr] following a period of cast immobilisation have been

implicated in the loss of muscular strength resultant to the period of disuse (Pathare et al.,

132

2005, 2008). Similarly, increases in the resting [Pi]:[PCr] ratio of patients with postpolio

residual paralysis, a condition which is characterised by decreased endurance capacity and

muscular weakness, is related to the severity of paralysis (Sharma et al., 2007). Increases

in intracellular [Pi] can inhibit force production via direct action on cross-bridge formation

or on sites in the excitation-contraction pathway and may play a key role in the

development of muscle fatigue (Westerblad et al., 2002). Thus the reduction in peak torque

and exercise tolerance observed in the present study following muscle damage, may be

related to the increased [Pi] and [Pi]:[PCr] which was observed not only at rest but also

during incremental exercise. The novel observation that the significant increases in resting

[Pi] and [Pi]:[PCr] at 48 h were maintained during the dynamic exercise test, is of particular

interest. The rate of increase in [Pi] during incremental exercise was not altered as a result

of the muscle damage. However the premature termination of the test at 48 h resulted in

there being no significant difference in end exercise [Pi] values. It might be tempting to

speculate that the limit to exercise tolerance was moderated by [Pi]. However wide inter-

subject variability was observed with the [Pi] values, thus we cannot reliably conclude that

[Pi] is indeed a limiting factor. Rather that end exercise [Pi] most likely makes an important

contribution to the reduced time to exhaustion experienced following muscle damaging

exercise.

Central fatigue factors including the production of inflammatory cytokines may be involved

in the reduced time to exhaustion following eccentric exercise observed herein. Carmichael

et al. (2005) have reported increases in brain IL-1β in areas responsible for movement,

motivation, perception of effort and pain, which have been associated with decreases in

treadmill runs to fatigue in mice. In human subjects, it has been proposed that the increased

133

sense of effort reported during dynamic exercise mediates time trial performance following

eccentric exercise (Marcora & Bosio 2007; Twist & Eston 2009) The duration of an

individual’s incremental exercise to ‘volitional exhaustion’ is regulated by a complex

interaction of central and peripheral fatigue factors. However, the decision to terminate

exercise is ultimately a conscious behavior based on the perception of alterations in sub-

conscious homeostatic control systems (St Clair Gibson et al., 2003). It is beyond the

scope of this study to determine whether central or peripheral factors make the greater

contribution to the accelerated fatigue development experienced with EIMD.

6.6 Conclusion

In conclusion, the results of this study suggest that the reduced exercise tolerance following

EIMD cannot be attributed to a greater rate of non-oxidative energy metabolism (as

inferred from the changes in [PCr] and pH). Although we cannot exclude an important role

for centrally-mediated fatigue, our results indicate that increases in resting [Pi], that are

maintained during exercise, may be a contributory factor to the reduced exercise tolerance

that is observed following EIMD.

The contents of this chapter form the basis of the following publication /presentation:

Publication

Davies RC, Eston RG, Poole DC, Rowlands AV, Dimenna F, Wilkerson DP, Twist C,

and Jones AM. The effect of eccentric exercise-induced muscle damage on the

dynamics of muscle oxygenation and pulmonary oxygen uptake. J Appl Physiol

(August 14, 2008). doi:10.1152/japplphysiol.90743.2008

Presentation

Davies R, Eston R, Poole D, Rowlands A, Dimenna F, Wilkerson D, Twist C, and

Jones A. The influence of exercise-induced muscle damage on pulmonary oxygen

uptake and muscle deoxygenation kinetics during high intensity exercise. 13th Annual

Congress of the European Congress of Sports Sciences, Book of Abstracts, edited by

Cabri J, Alves F, Araujo D, Barreiros J and Veloso A, Estoril, 2008, p630.

134

CHAPTER 7

THE EFFECT OF ECCENTRIC EXERCISE-INDUCED MUSCLE

DAMAGE ON THE DYNAMICS OF MUSCLE OXYGENATION AND

PULMONARY OXYGEN UPTAKE

135

7.1 Abstract

Unaccustomed eccentric exercise has a profound impact on muscle structure and function.

However, it is not known whether associated microvascular dysfunction disrupts the

matching of

O2 delivery ( Q˙ O2 ) to O2 utilisation ( V˙ O2 ). Near infra-red spectroscopy

(NIRS) was used to test the hypothesis that eccentric exercise-induced muscle damage

would

elevate

the

muscle Q˙ O2 : V˙ O2 ratio

during

severe

intensity exercise

whilst

preserving the speed of the V˙ O2 kinetics at exercise onset. Nine physically active men

completed ‘step’ tests to severe-intensity exercise from an unloaded baseline on a cycle

ergometer before and 48 h after eccentric exercise (100 squats with a load corresponding to

70%

body mass).

NIRS

and

breath-by-breath

pulmonary V˙ O2 were measured

continuously during the exercise tests and subsequently modelled using standard non-linear

regression

techniques. There

were

changes

phase

pulmonary V˙ O2 kinetics

following the onset of exercise (time constant, pre: 25 ± 4; post: 24 ± 2 s; amplitude, pre:

2.36 ± 0.23; post: 2.37 ± 0.23 L/min; all P>0.05). However, the primary (pre: 14 ± 3; post:

19 ± 3 s) and overall (pre: 16 ± 4; post: 21 ± 4 s) mean response time of the [HHb] response

was significantly slower following eccentric exercise (P<0.05). The slower [HHb] kinetics

observed following eccentric exercise is consistent with an increased

Q˙ O2 : V˙ O2 ratio

during transitions to severe-intensity exercise. We propose that unchanged primary phase

V˙ O2 kinetics are associated with an elevated Q˙ O2 : V˙ O2 ratio that preserves blood-

myocyte O2 flux.

136

7.2 Introduction

Unaccustomed eccentric exercise has a profound impact on muscle structure and function.

Following such exercise, myocytes demonstrate ultra-structural changes including

sarcomere disruption described as ‘popping’ (Morgan, 1990), ‘Z-band streaming’ (Fridén &

Lieber, 2001; Stupka et al., 2001) or ‘smearing’(Kano et al., 2005), and damage to t-

tubules, sarcoplasmic reticulum and sarcolemma (Fridén & Lieber, 2001 ). This disruption

leads to increased influx of extracellular Ca2+ into the sarcoplasm, leading to enhanced

proteolytic enzyme activity (Proske et al., 2004) and an accompanying inflammatory

response (Fielding et al., 1993). In addition, intramyocyte contents such as creatine kinase

and myoglobin are released into the bloodstream (Warren et al., 1999). These degenerative

changes are associated with delayed onset muscle soreness (DOMS) and a reduction in

maximal force generating capacity (Byrne et al., 2004; Clarkson, 1992; Cleak & Eston,

1992).

As myocyte degeneration is known to lead to decrements in maximal force production, any

associated damage to the microcirculation could potentially have an adverse affect on sub-

maximal locomotory activity such as running or cycling. Activities that require repetitive

low-force contractions rely on effective vascular function that ensures an adequate blood

and O2 supply to muscle. Accordingly, Kano et al. (2005) have reported substantial

microvascular dysfunction in rat spinotrapezius muscle following unaccustomed eccentric

exercise (downhill running). Specifically, these authors reported an increase in the

proportion of capillaries that did not support red blood cell (RBC) flux and an increase in

mean capillary diameter in resting muscle. Furthermore, an accelerated fall in

microvascular oxygen pressure was observed at the onset of electrically stimulated

137

contractions. Microcirculatory dysfunction such as this could conceivably lead to impaired

delivery and distribution of O2 within the capillary bed. Similarly, the matching of O2

delivery ( Q˙ O2 ) and O2 utilisation ( V˙ O2 ) at the onset of exercise might be

disturbed, thereby compromising blood-muscle O2 flux and, if sufficiently severe, slow the

kinetic adaptation of V˙ O2 at exercise onset (Kano et al., 2005). Although the compelling

weight of

evidence supports the premise that V˙ O2 kinetics in healthy individuals are not limited by

O2 delivery, per se, in disease conditions such as chronic heart failure (Richardson et al.,

2003) and Type II diabetes (Padilla et al., 2006) where vascular function and capillary

haemodynamics are impaired, V˙ O2 kinetics are slowed (Poole et al., 2005). It is possible

that microcirculatory dysfunction brought about by previous eccentric exercise could result

in the V˙ O2 kinetics of healthy individuals becoming slower due to a muscle O2 delivery

limitation. That is, the muscle damage could cause individuals to cross the so-called

‘tipping point’ beyond which reductions in muscle O2 availability begin to measurably

lengthen the time constant describing the phase II V˙ O2 response (Poole et al., 2008).

Near infrared spectroscopy (NIRS) facilitates the assessment of muscle (haemoglobin +

myoglobin) oxygenation and can thus be utilised to determine the dynamic balance between

Q˙ O2 and V˙ O2 following the onset of exercise. In particular, the deoxyhaemoglobin

(HHb) concentration ([HHb]) NIRS signal can be used to non-invasively estimate O2

extraction in the skeletal muscle microcirculation. Thus, the NIRS-derived [HHb] signal

would be expected to demonstrate a slower kinetic response at the onset of exercise if, as

138

anticipated, eccentric exercise does compromise blood-muscle O2 flux.

139

One recent investigation by Schneider et al. (2007) reported no change in V˙ O2 kinetics at

the onset of heavy intensity exercise 48 and 72 h following bench-stepping exercise

designed to incur damage. An explanation for this apparent paradox, i.e. normal

V˙ O2

kinetics in the face of severe muscle damage and impaired microvascular haemodynamics,

may be found in the work of Laaksonen et al. (2006) who reported that muscle damage

increased muscle blood flow during exercise. If microvascular function and therefore the

ability to match Q˙ O2 : V˙ O2

effectively is compromised following eccentrically exercised

muscle, it is possible that an elevation in the Q˙ O2 : V˙ O2 ratio would serve to raise

capillary O2 pressures and restore blood-tissue O2 flux in the face of capillary haemodynamic

derangements. Accordingly, the present investigation utilised NIRS to test the novel

hypothesis that eccentric exercise-induced muscle damage would elevate the muscle Q˙

O2 :

V˙ O2 ratio (as indicated by alterations in the [HHb] kinetics) during severe intensity

exercise and thus preserve the speed of the V˙ O2 kinetics at exercise onset.

7.3 Methods

Participants

Nine healthy men (mean ± S.D. age 22.7 ± 2.8 years; height 1.83 ± 0.06 m; mass 76.7 ± 7.0

kg), asymptomatic of illness and pre-existing injury, volunteered to participate in the study.

All were physically active but were not highly trained and had not undertaken any

resistance training of the lower limbs for at least six months prior to assessment.

Participants provided written informed consent to participate in the research which was

approved by the Ethics Committee of the School of Sport and Health Sciences at the

140

University of Exeter and conformed to the Declaration of Helsinki (See appendices B, C

141

and D for exemplar participant information sheet, participant consent form and ethical

approval certificate). The participants were requested not to take any anti-inflammatory

drugs for the duration of the study and to refrain from heavy exercise for 24 h prior to each

visit.

Procedures

All testing was performed on an electronically-braked cycle ergometer (Lode Excalibur

Sport, Groningen, Netherlands). The participants were instructed to report to the laboratory

at the same time of day (± 1 h) on five separate occasions within a period of 2-3 weeks

(Figure 7.1). On the first visit to the laboratory seat height and handlebar positions were

individually adjusted for comfort and the adjustments were recorded and replicated in

subsequent tests. The height and mass (SECA, UK) of each participant was also recorded.

Figure 7.1 Schematic overview of experimental procedures. V˙ O2 max,

Maximal oxygen uptake.

142

Participants then completed an incremental exercise test to volitional exhaustion to

determine maximal oxygen uptake ( V˙ O2 max) and gas exchange threshold (GET) and to

establish future work intensities. This entailed cycling at a self-selected pedal rate (between

70 and 90 rpm) for 4 minutes of baseline cycling at 0 W. The work rate was then increased

in a ramp fashion by 1 W every 2 s (i.e. 30 W.min−1) until the subject was unable to

continue. The

V˙ O2 max was defined as the highest 30-s mean value recorded before the

subject’s volitional termination of the test. The GET was determined independently by two

experienced reviewers using the V-slope method (Beaver et al., 1986). The work rate that

required 70% of the difference () between the GET and V˙ O2 peak (severe exercise) was

calculated, with account taken of the mean response time of the V˙ O2 adaptation to ramp

exercise (approximately 2/3 of the ramp rate i.e. minus 20 W) (Whipp, 1984). On the

second and fifth visits, pre and 48 h after the eccentric muscle damaging exercise

respectively, participants cycled at a self-selected pedal rate (between 70 and 90 rpm) for 4

minutes at 0 W, after which a warm-up phase at moderate intensity (~ 80% GET) was

applied for a further 6 minutes. After 2 minutes of passive rest, the subjects pedalled for 4

minutes of baseline cycling at 0 W after which the severe work rate was abruptly applied

for 6 minutes. This test was repeated after a rest period of 2 hours. In the second test, the

severe exercise bout was continued until the subject volitionally terminated the test at

exhaustion.

Eccentric exercise

In order to provoke muscle damage, participants performed 100 (Smith) squats as 10 sets of

10 repetitions. The load on the bar was calculated to correspond to 70% of each

143

participant’s body mass. For further details of these procedures please refer to Chapter 3.

144

Measurements

Markers of muscle damage

Markers of muscle damage (muscle soreness and isokinetic peak torque) were measured

immediately before and then 30 minutes and 48 hours after eccentric exercise. In addition

plasma creatine kinase (CK) activity was assessed immediately before and then 30 minutes,

24 and 48 hours after eccentric exercise. Due to availability at 24 h, only 8 participants

were measured for CK activity.

Assessment of muscle damage

All indicators of muscle damage, perceived muscle soreness (using a 0-10 visual analogue

scale (VAS)), creatine kinase (CK) activity and isokinetic peak torque (30 deg.s-1), were

measured in the order listed, immediately before, and 24 and 48 h after performing the

eccentric, muscle-damaging exercise protocol. For further details of these procedures

please refer to Chapter 3.

Exercise test measures

Pulmonary gas exchange and ventilation were measured breath-by-breath throughout all

tests with participants wearing a nose clip and breathing through a low-dead-space, low-

resistance mouthpiece via an online gas analysis system (Cortex MetaMax 3B, Biophysik,

Leipzig, Germany). The system was calibrated prior to every test in accordance with

manufacturer’s guidelines against known concentrations of cylinder gases (5% oxygen,

15% carbon dioxide) and a 3 l calibration syringe (Hans Rudolph, Kansas City, MO). The

145

V˙ O2data gathered from the pre- and post- exercise tests were subsequently modelled to

provide estimates of the V˙ O2 kinetic parameters (see Modelling of V˙O2 and [HHb] data).

Heart rate (HR), blood lactate concentration ([La]) and Ratings of Perceived Exertion

(RPE) (Borg, 1998) were recorded at 2 min, 4 min and at the end of exercise. HR and [La]

measures were also recorded during baseline cycling. HR was monitored using a wireless

chest strap telemetry system (Polar Electro T31, Kempele, Finland) and measured

continuously via a link to the Cortex gas analysis system. Fingertip blood samples were

collected and analysed for [La] using an YSI 2300 STAT plus analyzer (Yellow Springs,

Ohio, USA). Participants were familiarized with Borg’s 6-20 RPE Scale and provided with

standardised instructions on how to employ the scale (Borg, 1998). Participants were

encouraged to focus on their overall perception of exertion when reporting their RPE.

Near-infrared spectroscopy (NIRS)

Oxygenation profiles of the right vastus lateralis muscle were recorded in all exercise tests

using a continuous wave near-infrared spectrometer (NIRS) (Hamamatsu NIRO 300,

Hamamatsu Photonics KK, Japan) (Figure 7.2). The system monitored concentration

changes in oxyhaemoglobin (HbO2) and deoxyhaemoglobin (HHb) which were calculated

from the light attenuation change by utilising the modified Beer-Lambert law. The HHb

concentration ([HHb]) signal obtained from the NIRS was regarded as being relatively

insensitive to blood volume changes during exercise and thus reflected the balance between

the delivery and utilisation of oxygen (Ferrari et al., 1997).

146

Figure 7.2 Measurement principle and probe structure of the NIRO 300 (Hamamatsu,

Hamamatsu Photonics KK, Japan)

Pulsed light was emitted at 1 s intervals from the emission probe at four different

wavelengths (775, 810, 850 and 910nm) and was detected, as a function of distance, using a

three segment photodiode detection probe that received NIRS signals at 2 Hz. The probes

were housed in the black silicone holder provided. The inter-optode spacing between

emitter and receiver was 4 cm, and the penetration depth was approximately half of the

distance between the emitter and the receiver, i.e. 2 cm. Prior to placement on the right

vastus lateralis, the site was shaved and cleaned using an alcohol swab. The NIRO300

system was then calibrated and the probe holder secured by means of a double-sided

adhesive sheet ~12 cm above the lateral epicondyle of the right leg, with the location

marked using an indelible marker pen to enable reproduction of the probe positions in

subsequent tests (48 h). The thigh with attached probe holder was then wrapped in a dark-

coloured, elastic bandage to further secure the probes and to eliminate ambient light that

might contaminate the NIRS signal (Figure 7.3)

147

Figure 7.3. Attachment of the NIRS probes. The NIRS probe holder is secured by

means of a double-sided adhesive (a). The thigh with attached NIRS probe holder is

wrapped in an elastic bandage (b).

The NIRS data gathered represented relative concentration changes in the haemoglobin

chromophores and were, therefore, not representative of absolute tissue O2 values. As

[HHb] was measured as a change from baseline values the probe gain was zero set prior to

testing with the subject at rest in a seated position. Differences in the thickness of the

overlying adipose tissue may influence the amplitude of the NIRS signal. However, the

same subjects were employed pre- and post eccentric exercise and the probe positions were

rigorously maintained for each subject in each test, thus no correction for inter-site

adiposity was necessary. Following exercise testing the data were downloaded and the

resulting text files stored for subsequent analysis.

(a) (b)

148

Modelling of V˙O2 and [HHb] data

The breath-by-breath data from each exercise test were filtered manually to remove

outlying breaths, defined as breaths ± 3 SD from the adjacent five breaths. The data for

each individual were then interpolated to provide 1 s values and the two data sets from each

of the pre- and post- exercise tests were time-aligned and averaged. The first 20 s of data

after the onset of exercise (the Phase I response) were deleted and a biexponential model

was used to analyze the V˙ O2 responses to severe exercise, as described by the following

equation:

V˙ O2 (t) = V˙ O2 baseline

+ Ap[1 – e

Tdp)/

τp

] (phase 2 / primary component)

+ As[1 – e

–

Tds)/

τs

] (phase 3 / slow component)

where t is time;

V˙ O2 baseline is baseline

V˙ O2 ; Ap and As are the primary and slow

component amplitude, respectively; Tdp and Tds are the primary and slow component time

delays, respectively; and τp and τs are the time constants of the primary and slow

components respectively.

The parameters

of the

model

were determined

using

nonlinear least squares algorithm. In the equations above, V˙ O2 (t) represents the absolute

V˙ O2

at a given time t, and V˙ O2 baseline represents the average V˙ O2

through the baseline

cycling period. Because the time to exhaustion was not identical in the first and second

bouts of severe exercise, we fitted the data 1) to the end of exercise in both bouts and 2) to

the same point in time (given by the time to exhaustion in the shortest bout). The primary

149

component “gain” (i.e., Ap/∆WR) was calculated from the projected asymptotic V˙ O2 . In

addition, the “actual” gain attained at the end of exercise was calculated.

To provide information on the effect of eccentric muscle-damaging exercise on the

dynamics of muscle oxygenation, we also modelled the ∆[HHb] response to severe

exercise. The NIRS-derived [HHb] data were time-aligned and averaged to provide a single

response for each subject pre- and post- eccentric exercise. The time delay before an

increase in [HHb] after exercise onset was determined as the first point greater than one

standard deviation above the mean of the baseline (DeLorey et al., 2003). [HHb] data were

then fitted with a bi-exponential model similar to that described by the equation above, with

the exception that the fitting window started at the onset of exercise (i.e., at time 0).

Subsequently, [HHb] data were fitted with a mono-exponential model from the onset of

exercise to the time point representing the interface of the primary and slow component to

determine the rate of adaptation of muscle deoxygenation during the primary phase

(MRT1). In addition, the [HHb] dynamics for the entire response were modelled with a

similar mono-exponential function (MRTt).

Statistical analysis

Changes in the markers of muscle damage (peak torque, soreness and CK activity) were

analysed using a series of one-way repeated measures (RM) ANOVA. All data were

checked for assumptions of normality. As the CK activity data were found not to be

normally distributed, the values were log-transformed prior to statistical analysis (Twist &

Eston, 2005) (see appendix H).

150

Following transformation CK activity data were normally distributed. Changes in HR,

RPE, [La] and ventilation were analysed using separate 2-way RM ANOVAs (test x time).

Assumptions of sphericity were evaluated using Mauchly’s test. Where sphericity was

violated (P< 0.05) the Greenhouse-Geisser (GG) correction factor was applied. Post-hoc

Tukey tests modified for repeated measures (Stevens, 2002) were run to determine where

significant

differences

occurred.

Paired

t-tests

were

used

determine

significant

differences in time-to-exhaustion and the V˙

and [HHb] kinetic responses to severe

intensity exercise before and after eccentric exercise. All data were analysed using the

statistical software package SPSS for Windows (version 13). Statistical significance was set

at 0.05.

7.4 Results

Markers of muscle damage

The eccentric exercise was effective in provoking significant changes in all markers of

muscle damage. Table 7.1 shows changes in isokinetic peak torque, perceived muscle

soreness and plasma CK activity before and at 24 h and 48 h post eccentric exercise.

151

Table 7.1 Changes in markers of muscle damage. Mean ± SD values before (pre)

and at 24 h and 48 h after eccentric exercise.

Measured variable pre 24 h post 48 h post

Peak Torque (N.m) 30 deg.s-1 287 ± 39 227 ± 44* 228 ± 60*

Soreness 0.6 ± 0.5 6.4 ± 1.8* 7.1 ± 1.5*

CK activity (U/L) 172 ± 123 740 ± 666* 373 ± 208

Values are means ± SD before (pre) and 24 h and 48 h after eccentric exercise (post).

Soreness, visual

analogue scale 0-10. CK, creatine kinase. *significantly different (P < 0.05) from pre value

Isokinetic peak torque (30 deg.s-1) decreased by 21% at 24 h post-eccentric exercise and

remained depressed at 48 h (F (2,16) = 21.85 p < 0.001). Significant soreness was reported

24 h after eccentric exercise with the highest values at 48 h (F (2,14) = 80.50 p < 0.001).

Plasma CK activity increased after eccentric exercise, with the highest activity observed at

24 h (F (2,16) = 17.15 p < 0.001). Changes in markers of muscle damage were detected in all

subjects, although considerable inter-subject variability was observed. Peak decrements in

isokinetic peak torque (30 deg.s-1) ranged from 12 - 44%, and peak increases in soreness

and plasma CK activity ranged from 53 - 95% and 146 - 1176%, respectively.

Response to severe intensity exercise

Table 7.2 shows the V˙ O2 responses to severe intensity exercise. There were no changes in

the phase II V˙ O2 kinetics following eccentric exercise, nor was there a change in the slow

component time delay (TD) (P > 0.05). However the amplitude of the slow component was

significantly reduced (t (8) = 3.84, P < 0.05) and the overall mean response time (MRT) was

significantly faster (t (8) = 4.01, P < 0.05) following eccentric exercise. A significantly

shorter time to exhaustion was observed in the 2nd bout of severe intensity exercise (pre:

152

7:24 min:s (± 2:41) post 6:14 min:s (± 2:46)) (t (8) = 2.58, P < 0.05). The V˙ O2 response of

a representative subject is illustrated in Figure 7.4.

Table 7.2

Pulmonary O2 uptake responses to severe intensity exercise before and after

eccentric muscle damaging exercise.

Pre 48 h post

Primary component

TDp (s) 12 ± 4 12 ± 2

p (s) 25 ± 4 24 ± 3

Ap V˙ O2 (L·min-1)2.36 ± 0.23 2.37 ± 0.23

Gain (mL·min-1·W-1)9.02 ± 0.45 9.04 ± 0.52

Slow component

TD2 (s) 102 ± 19 107 ± 29

A2ref V˙ O2 (L·min-1)0.63 ± 0.33 0.37 ± 0.17*

A2end V˙ O2 (L·min-1)0.67 ± 0.33 0.38 ± 0.17*

Overall response

MRT

ref

61 ± 15 51 ± 9*

MRT

end

64 ± 15 51 ± 9*

Peak V˙ O2 (L·min-1)3.89 ± 0.44 3.70 ± 0.36

Values are means ± SD. TDp, p. Ap and gain are the time delay, time constant, amplitude and increase in

V˙ O2 per unit increase in work rate for phase II kinetics respectively. TD2, A2ref and A2end are the time delay,

amplitude to 6 mins and amplitude to end of exercise for the slow component, respectively. MRTend and

MRTref are the mean response times fitted to the end of exercise in both bouts and to the same point in time

(given by the time to exhaustion in the shortest bout), respectively. Peak V˙ O2 tended to be lower post-

eccentric exercise but the difference was not statistically significant. Significant difference (P < 0.05) from

pre value.

153

Figure 7.4 A representative subject’s V˙ O2 response to severe cycle exercise pre-

(●) and 48 h post- (○) eccentric exercise. The vertical line represents the transition from

‘unloaded’ to ‘loaded’ cycling.

There was a significant increase in RPE values reported during severe intensity exercise

following eccentric exercise (F (1,8) = 6.7 P < 0.05). However there were no significant

differences in the blood lactate or heart rate responses before and after eccentric exercise

(Table 7.3) (P > 0.05). In addition, there was a significant increase in the ventilatory

equivalent for O2 (/ V˙ O2 ) following eccentric exercise (pre: 29.3 (± 3.5) post: 32.6 (±

5.2))

(F (1,8) = 7.45, P< 0.05).

154

Table 7.3 Ratings of Perceived Exertion (RPE), Heart Rate (HR) and blood lactate

concentration ([La]) during severe intensity exercise pre- and 48 h post-eccentric

exercise.

baseline min

2 min

4 end

exercise

pre 48 h pre 48 h pre 48 h pre 48 h

RPE* NA NA 15 ± 1 16 ± 1 17 ± 1 18 ± 2 19 ± 1 19 ± 1

HR (b.min-1)83 ± 9 86 ± 6 159 ± 8 163 ±11 174 ±11 176 ±11 184 ± 9 181 ±11

[La] (mmol.l-1)0.9 ± 0.5 1.0 ± 0.4 3.5 ± 1.0 3.8 ± 1.0 6.9 ± 1.3 6.8 ± 1.8 8.4 ± 1.4 8.7 ± 1.8

Values are means ± SD. RPE, ratings of perceived exertion based on Borg’s 6-20 scale (Borg, 1998); HR,

heart rate; [La], blood lactate concentration; NA, not applicable.

* Significant main effect for time (P < 0.05).

There was also a significant interaction of time x test (F (9,72) = 3.15, P< 0.05) on V˙E / V˙ O2 .

Post-hoc Tukey tests indicated that

V˙

/ V˙ O2 was significantly greater post eccentric

exercise for the last 70% of exercise with mean values rising from 25.3 to 36.0 (pre ) and

from 26.7 to 39.9 (post) (P< 0.05).

The results of the kinetic response of [HHb] to severe intensity exercise pre- and 48 h post-

eccentric exercise are shown in Table 7.4. Most importantly, with respect to our

experimental hypothesis, both the [HHb] MRT1 and MRTt were significantly slower

following eccentric exercise (P < 0.05). There was no significant correlation between any

of the markers of muscle damage and indices of muscle oxygenation in the post damage

condition (P > 0.05). The [HHb] response of a representative subject is illustrated in Figure

7.5.

155

Table 7.4 [HHb] response to severe intensity constant-load exercise pre- and 48 h

post-eccentric exercise.

Pre 48 h

MRT1 (s) 14 ± 3 19 ± 3*

Primary Amp (%) 91 ± 8 88 ± 8

Primary Amp (A.U.) 309 ± 102 297 ± 72

MRTt (s) 16 ± 4 21 ± 4*

SC Amp (%) 9 ± 8 12 ± 8

SC Amp (A.U.) 33 ± 25 41 ± 25

Values are means ± SD. [HHb], deoxyhaemoglobin concentration; MRT1 and MRTt are the mean response

time of the primary phase (the rate of adaptation of muscle deoxygenation during the primary phase) and the

overall response (the rate of adaptation of muscle deoxygenation for the entire response), respectively.

Primary Amp is the change in [HHb] during the initial, fast increase expressed as a percentage of the overall

response (%) and as arbitrary units (A.U.). SC Amp is the change in [HHb] during the slow component (the

additional increase in [HHb] which develops slowly when exercise intensity is above the gas exchange

threshold) expressed as a percentage of the overall response (%) and as arbitrary units (A.U.)

*48 h post value significantly slower than pre value (P < 0.05).

Figure 7.5 Deoxygenated Hb ([HHb]) response to severe cycle exercise pre- (●) and 48

h post- (○) eccentric exercise in a representative subject. The vertical line represents the

transition from unloaded to loaded cycling. Note the slower overall [HHb] kinetics

following eccentric exercise (the slower rate of adaptation of muscle deoxygenation or

fractional O2 extraction, for the entire response),. AU, arbitrary units.

156

The altered Q˙ O2 :

V˙ O2

balance is most pronounced during the initial response following

the onset of severe intensity exercise (Figure 7.6), with the greatest mean difference

observed over the first 5-20 s (Figure 7.7). There were no significant differences in the pre-

and post-conditions between the amplitude of the response in either the primary phase or

slow component (P > 0.05). Similarly there was no difference between the total

haemoglobin responses in the two conditions (Figure 7.8).

Figure 7.6

Mean initial [HHb] response to severe cycle exercise pre- (●) and 48 h post-

(○) eccentric exercise. The vertical line represents the transition from ‘unloaded’ to

‘loaded’ cycling. The data are normalized for amplitude of the response at baseline. Values

are mean (± SE). AU, arbitrary units.

157

Figure 7.7

Mean difference in the initial [HHb] response to severe intensity exercise

pre- and 48 h post eccentric exercise (i.e. pre- minus post-eccentric exercise data). AU,

arbitrary units.

7.5 Discussion

The principal original finding of this investigation is that eccentric, muscle-damaging

exercise results in a slowing of muscle [HHb] kinetics without altering pulmonary V˙ O2

kinetics during high-intensity cycle exercise in humans. We interpret the slower [HHb]

kinetics, in the face of unchanged pulmonary V˙ O2 , to be consequent to a local elevation

the Q˙ O2 :

V˙ O2

ratio. The observation that the [HHb] kinetics were over 30% slower 48 h

after the performance of eccentric exercise, suggests that the matching of Q˙ O2

and

V˙ O2

158

was profoundly altered as a consequence of the intervention.

159

Figure 7.8

Total haemoglobin response to severe cycle exercise pre- (●) and 48 h post-

(○) eccentric exercise. AU, arbitrary units. Changes in total haemoglobin were not

different between conditions such that changes in [HHb] provided valid measures of muscle

deoxygenation (fractional O2 extraction). Increased blood flow, such as has been observed

by Laaksonen et al. (2006) could compensate for the decreased proportion of capillaries

supporting RBC flow such as has been observed by Kano et al. (2005) and therefore

maintain the total haemoglobin response in the area of interrogation.

The dynamic balance between O2 delivery and O2 utilisation has been keenly debated with

regard

possible

limitations

muscle V˙

kinetics

(Poole

al.,

2008).

During

transitions to exercise intensities below GET, the compelling weight of evidence supports

the premise that metabolic inertia is the principal limitation to muscle O2 uptake (Bangsbo

et al., 2000; Grassi et al., 1998, 1996). However, for transitions to exercise intensities above

GET, the delivery of O2 may exert an additional modest constraint on muscle V˙ O2

kinetics

160

(Grassi et al., 2000, 2003; Tschakovsky & Hughson, 1999). The present data indicate that

following eccentric exercise-induced muscle damage, V˙

kinetics are preserved by an

161

increase in local muscle blood flow which is presumably necessary to compensate for

microcirculatory dysfunction (Kano et al., 2005). Increased blood flow, such as has been

observed by Laaksonen et al. (2006) following eccentric exercise, could compensate for the

decreased proportion of capillaries supporting RBC flow such as has been observed by

Kano et al. (2005). In support of this thesis, we observed no change in the total

haemoglobin response, indicative of local muscle blood flow in the area of interrogation.

Thus, the changes in the [HHb] response provided valid measures of muscle deoxygenation

(fractional O2 extraction). These data therefore imply that the subjects were operating to

the right of the ‘tipping point’ (Poole et al., 2008) during the exercise bout, such that

compensatory changes in local muscle blood flow were able to prevent a measurable

slowing of V˙ O2 kinetics following muscle damage.

The squatting protocol employed herein (Byrne & Eston, 2002a, 2002b), was effective in

inducing muscle damage as indicated by the significant decreases in peak torque and

increases in plasma CK activity. Despite this, there was no significant change in phase II

V˙ O2 kinetics following eccentric exercise. These data are consistent with Schneider et al.

(2007) who reported that the V˙ O2 and HR responses to heavy-intensity cycling exercise

were unaffected by prior eccentric exercise and surmised that a moderate degree of muscle

damage did not impair O2 delivery to the active muscle or alter the

Q˙ O2 / V˙ O2 ratio.

However, indices of muscle oxygenation were not measured by these authors (60). Our

findings

contrast

with

those

Ahmadi

al.

(2008),

who

reported

faster

oxygen

desaturation (analogous to a lower Q˙ O2 / V˙ O2 ratio) during isometric contractions at 30%,

50% and 80% MVC following exhaustive downhill walking. However, differences in the

162

experimental protocol and exercise modalities employed make it very difficult to compare

the two studies. The high intramuscular pressure generated during isometric contractions is

known to impede muscle blood flow and this, or an increase in the energy demand of

contraction, might account for the faster oxygen saturation reported by Ahmadi et al.

(2008).

The experimental protocol used in the present study differs fundamentally from that

employed by Kano et al. (2005). These authors electrically induced twitch muscle

contractions (1 Hz, 3–5 V, 2-ms pulse duration) in rat spinotrapezius muscle, whereas in

our study, human subjects performed dynamic high-intensity cycle-exercise transitions.

Whilst taking these differences into consideration, it is important to note that rat

spinotrapezius muscle does provide a highly acceptable, comparative model for the analysis

of human microvascular and myocyte damage as it exhibits a fibre composition (Delp &

Duan, 1996) and oxidative capacity (Leek et al., 2001) that closely resemble that of the

human quadriceps. Thus the disruption observed in rat microvasculature subsequent to

eccentric exercise would, most likely, also be present in the damaged muscle of subjects

interrogated herein. Kano et al. (2005) reported disrupted capillary geometry and

substantial microvascular dysfunction following eccentric exercise. Specifically, an

increase in the capillary luminal area of damaged muscle was reported which resulted in a

decreased PO2mv at the onset of electrically stimulated contractions. In addition a 27–34%

increase in non-RBC-flowing capillaries was found 1–3 days after a single bout of eccentric

exercise.

163

Such structural and functional alterations to the microvasculature might act to alter the

matching of Q˙ O2 to

V˙ O2 both spatially and temporally with respect to tissue energetic

requirements.

Specifically,

the lower

[HHb]

observed

any

given V˙ O2 across the

transition post-eccentric exercise suggests that achievement of the requisite blood-myocyte

O2 flux might demand a higher microvascular O2 driving pressure. Blood-muscle O2 flux is

primarily determined by the number of erythrocytes lying adjacent to active myocytes at

any given time (Federspiel & Popel, 1986; Groebe & Thews, 1990). A decrease in the

proportion of capillaries supporting RBC flow would lead to a reduction in blood-muscle

O2 flux. Similarly, an increase in the diameter of free-flowing capillaries would increase the

carrier-free diffusion distance and lead to a reduction in O2 diffusing capacity.

Fick’s law of diffusion states that:

V˙O2 m = DO2 (PO2mv

–

PO2 intramyocyte)

where DO2 is the diffusing capacity for O2 and PO2mv is the microvascular O2 pressure.

Such reductions to DO2 would be expected to negatively impact V˙ O2 particularly in the

presence of concomitant alterations to PO2mv similar to those reported by Kano et al.

(2005). The O2 pressure gradient from blood to myocyte which drives O2

diffusion is

determined principally by alterations of PO2mv (Poole et al., 2006; Poole & Ferreira,

2007). Thus the accelerated fall of PO2mv observed by Kano and colleagues during the first

20–40 s of electrically stimulated muscle contractions would be expected to have a

profound influence on the muscle O2 diffusing capacity and result in a slowing of V˙ O2

164

kinetics (Behnke et al., 2004, 2007, 2006). However, the PO2mv herein (as judged by the

[HHb] response) appeared to be elevated during the dynamic rest-exercise transitions post-

eccentric exercise and the V˙ O2 kinetic response remained unchanged.

A given V˙ O2

is achieved through the interaction of O2 delivery ( Q˙ O2 ) and O2 diffusing

properties (Behnke et al., 2003). Additionally, changes in pulmonary V˙ O2 across the rest-

exercise transition are know to directly reflect leg V˙ O2 during cycling exercise (Poole et

al., 1991), and demonstrate a good approximation of the muscle V˙ O2 kinetics (Grassi et al.,

1996). Therefore, the elevated

Q˙ O2 : V˙ O2 ratio evidenced by the slower [HHb] kinetic

response (i.e. lower [HHb] at a given V˙ O2 across the exercise transition) must, in this

instance, be due to compensatory changes to O2 delivery (see Appendix J). Pertinent to this

issue, increased blood flow (and therefore increased O2 delivery) has been observed by

Laaksonen et al. (2006) who found that blood flow to the exercising Quadriceps femoris

was elevated by 25% after a prior bout of exhaustive eccentric exercise. Importantly, they

also reported that

V˙ O2 remained unchanged and suggested that the altered

Q˙ O2 : V˙ O2

balance observed may have been due to impaired oxygen extraction. Thus, following

eccentric muscle-damaging exercise, adaptive, compensatory mechanisms may act to

elevate PO2mv across the rest-exercise transition, preserving blood-myocyte O2 flux as

indicated by the unchanged phase II V˙ O2 kinetics.

The higher ratings of perceived exertion reported for a given exercise intensity (70 %∆)

following eccentric exercise may be due, in part, to the enhanced ventilatory response

165

which accompanies the increased muscle soreness. It has been proposed that an altered

166

sense of effort may be part of a central protective mechanism whereby neural inhibition

serves to reduce force generation in order to prevent further injury (Kyrolainen et al., 2000;

Michaut et al., 2002; Proske et al., 2004). Additionally, Hotta et al. (2006) suggested that

changes in neural factors contribute not only to alterations in force generation but also to an

enhanced ventilatory response. Group III and IV afferent fibres located in and around the

blood vessels of exercising muscle are involved in modulating the ventilatory response.

Distension of these blood vessels provokes a discharge from the afferent fibres that leads to

an increase in ventilation (Haouzi et al., 2004, 1999). Thus, neural monitoring of peripheral

vascular events might account, in part, for the enhanced / V˙ O2 observed in this study if

there were alterations to the microvasculature as a result of eccentric exercise. Pertinent to

this issue, in studies employing male Wistar rats, Kano et al. (2004) have reported changes

in the capillary lumen shape (luminal ellipticity) that increase the luminal cross-sectional

area by up to 62% following eccentric exercise. Similar increases in the cross-sectional area

of the microvessels in the muscles recruited in the present investigation might serve to

augment the ventilatory response via neural modulation.

It has been proposed that, following eccentric exercise, alterations to motor unit recruitment

patterns arise in order to meet the energetic demands of a given work rate (Clarkson, 1992).

Reports of elevated blood lactate concentration following eccentric exercise have been

attributed to the additional recruitment of type II fibres and a concomitant rise in the rate of

glygogenolysis (Chen et al., 2007b; Gleeson et al., 1998). However, Chen (2003) reported

that neural adaptation to motor unit activation patterns occurred after a single bout of

eccentric exercise, such that additional type I motor units were recruited. Additional fibre

recruitment has been implicated in the development of the V˙ O2 slow component in high-

167

intensity exercise (Krustrup et al., 2004). Specifically, it has been proposed that the

development of a

V˙ O2 slow component may be related, in part, to the recruitment of

additional type II fibres (Barstow et al., 1996; Pringle et al., 2003; Whipp, 1994). The

reduction in the amplitude of the V˙ O2 slow component observed herein may therefore be

indicative of an altered motor unit recruitment pattern consequent to muscle damage

(Pringle et al., 2003). However, the unchanged primary V˙ O2 response and the unchanged

blood lactate concentration observed are arguably not in keeping with this suggestion.

Additionally, full expression of the slow component may have been impeded as a result of

muscular fatigue and a reduction in maximal work capacity brought about by the effects of

eccentric exercise. Such impediments to performance have previously been reported to

result in a shorter time to exhaustion in mice (Carmichael et al., 2005, 2006) and a reduced

time trial running distance in human subjects (Marcora & Bosio, 2007). Accordingly, the

reduction in the V˙ O2 slow component observed post-eccentric exercise may be attributed

to a shorter time to exhaustion and a tendency for a lower V˙ O2 peak to be achieved in this

condition.

Experimental considerations

As mentioned earlier, our findings contrast with those of Kano et al. (2005) due to

fundamental differences in the muscle activation processes employed rather than species

variation. We studied dynamic exercise transitions to severe intensity (70 %∆) cycle

exercise, whereas Kano and colleagues utilised a set rate of electrical stimulation (1 Hz, 3–

5 V, 2-ms pulse duration) to induce muscle contractions. Electrical stimulation induces

recruitment of all fibres, whilst voluntary exercise recruits specific fibres and fibre types

168

dependant on exercise intensity and duration (Kindig et al., 2002). Thus, the muscle

activation and attendant fibre recruitment patterns studied herein present a more

ecologically valid model for investigation. As such, this study may provide a more realistic

insight into the effects of eccentric muscle-damaging exercise on functional human

performance.

NIRS is an established technique for the measurement of muscle oxygenation (e.g. Ferrari

et al., 2004; Jones et al., 2006). However, the reliability and reproducibility of the NIRS-

derived [HHb] signal is dependant on the precise placement of the optodes. In the present

study, optode location was marked on each individual subject during the first visit to the

laboratory and placement was carefully reproduced on subsequent visits. Koga et al. (2007)

have revealed the presence of significant heterogeneity with respect to the dynamics of

muscle oxygenation within the quadriceps muscles of healthy subjects following the onset

of exercise. These findings are not surprising given that muscle blood flow, motor unit

distribution and recruitment and consequently vascular responses are known to be

heterogeneous within and across muscles. However, it is important to recognise that the

NIRS data reported herein is representative of changes within the superficial muscle area

under interrogation only and as such may not be representative of the entire muscle mass

affected by eccentric exercise.

169

7.6 Conclusion

In conclusion, the present investigation suggests that eccentric, muscle-damaging exercise

alters the matching of Q˙ O2 and V˙ O2 during severe intensity exercise. Specifically, across

the rapid metabolic transition following the onset of exercise, for a given V˙ O2 , the [HHb]

signal is reduced. We propose that structural and possibly functional alterations to the

microvasculature act to increase the Q˙ O2 : V˙ O2 ratio both spatially and temporally with

respect

tissue

energetic

requirements.

Accordingly,

following

eccentric

muscle-

damaging exercise, compensatory mechanisms act to elevate the microvascular driving

pressure for blood-myocyte O2 flux, enabling a preservation of the kinetics of V˙ O2 across

the rest-to-exercise transition.

170

CHAPTER 8

CONCLUSIONS

171

8.1 Main Findings

8.1.1 The ventilatory response to dynamic exercise with EIMD

Previous investigations have reported that the ventilatory response to running exercise is

unchanged following EIMD (Paschalis et al., 2005; Scott et al., 2003; Marcora and Bosio,

2007). Other investigations that reported an increased ventilatory response and an elevated

blood [La] response during running attributed the changes to altered lower limb kinematics

(Braun & Dutto, 2003; Chen et al., 2007b, 2008). During cycle exercise, where the

potentially confounding influence of altered kinematics is avoided, increases in both

ventilation and blood [La] have been reported (Gleeson et al., 1995; Schneider et al., 2007).

Prior to the studies which comprise this thesis being undertaken, Schneider et al. (2007),

using an exercise intensity of 40%∆, provided the only example of an investigation using a

specific exercise domain to study the influence of EIMD on physiological responses to

dynamic exercise.

The findings of studies 1 (Chapter 4) and 4 (Chapter 7) demonstrated that the ventilatory

response to cycle exercise was elevated 48 h after eccentric exercise (100 squats with a load

corresponding to 70% of body mass). Importantly, ventilation was increased not only

during severe intensity (70% ∆) cycle exercise but also during moderate intensity (80%

GET) cycle exercise without any significant change in the blood [La] response. Previously,

it had been assumed that an increase in [La] contributed to an augmented ventilatory

response. However, our findings, in particular the observation that the ventilatory response

following EIMD is higher during exercise below GET, suggest that the altered exercise

response may not be due to changes in metabolic factors. Rather, we propose that neural

monitoring of peripheral vascular and local muscular events may, in part, account for the

172

augmented ventilatory response observed. The mechanisms involved in ventilatory control

during exercise are complex and involve a combination of central command and afferent

feedback. However, the structural and functional changes that characterise EIMD are

understood to augment discharge from group III and IV afferents. These changes rather

than elevated blood [La] which is not an obligatory consequence of EIMD, most probably

contribute to the greater ventilatory response to dynamic cycle exercise evoked by EIMD.

8.1.2 The Gas Exchange Threshold (GET) and EIMD

During dynamic incremental exercise protocols, identification of the GET provides the

basis for the non-invasive estimation of the lactate threshold (Tlac) (Beaver et al., 1986;

Caiozzo et al., 1982; Wasserman et al. 1973, 1990). However, the ventilatory and lactate

responses to incremental exercise may be dissociated by various experimental and clinical

conditions (e.g. Poole & Gaesser, 1985; Hughes et al., 1982; Hagberg et al., 1982). It was

our contention that EIMD was likely to exacerbate the ventilatory response to

incremental/ramp exercise and, in so doing, potentially dissociate the ventilatory, gas

exchange and lactate responses.

The findings of study 2 (Chapter 5) demonstrated that 48 h after eccentric exercise GET

occurred at a lower work rate (pre, 136 ± 27 W; post, 105 ± 19 W, P< 0.05) and V˙ O2 (pre,

1.58 + 0.26; post, 1.41 + 0.14 l.min-1, P< 0.05). However, the lactate threshold occurred at

a similar work rate (pre, 161 ± 19 W; post, 158 ± 22 W, P> 0.05) and V˙ O2 (pre, 1.90 ±

0.20 l.min-1; post, 1.88 ± 0.15 l.min-1, P > 0.05) after eccentric exercise. These findings

demonstrated that EIMD dissociates the ventilatory response to incremental exercise from

the blood [La] response. Thus, these findings provide further evidence to support the thesis

173

that ventilation may be controlled by additional or altered neurogenic stimuli following

eccentric exercise.

8.1.3 The perception of exertion during dynamic exercise with EIMD

Irrespective of the mode of damage, the exercise intensity, or exercise protocol employed,

eccentric, muscle-damaging exercise is known to evoke an increased sense of effort

(Gleeson et al., 1995; Scott et al., 2003; Marcora & Bosio, 2006; Twist & Eston, 2009).

The cues which inform the perceptual response to exercise may arise from central or

peripheral sensations, with the influence of central (cardiorespiratory) cues of lesser

importance than that of local (muscle) sensations, particularly at lower exercise intensities

(Mihevic, 1981; Hampson et al., 2001). As such, the perception of exertion reported during

exercise with EIMD may be differentially influenced by central and peripheral cues

dependent on the exercise intensity.

During ramp/incremental cycle exercise in study 3 (Chapter 5) participants reported a

higher perception of exertion at 48 h, with an increase in reported RPE of 7% observed at

the V˙

value of the pre-eccentric exercise GET. In studies 1 and 4 (Chapters 4 and 7)

participants reported higher ratings of perceived exertion (RPE) during constant-load,

severe intensity (70%∆) cycle exercise 48 h after eccentric exercise, although RPE

appeared to be unchanged during moderate (80% GET) exercise. It was of interest that the

increased muscle pain and the elevated ventilatory response experienced by participants at

48 h did not significantly influence the perception of exertion during moderate intensity

exercise. However, during severe intensity exercise, where the central, ventilatory cues may

have played a more influential role in informing the perception of exertion, RPE was

174

significantly higher. In study 1 (Chapter 4) minute-by-minute differences in RPE values

reported during severe exercise to volitional exhaustion before and 48 h after eccentric

exercise were eliminated when expressed as a percentage of total exercise duration. This

observation provided further evidence to support the proposition that there is a scalar-linear

relationship between the rating of perceived exertion and exercise duration. Furthermore,

differences in ventilation were also eliminated when expressed as a proportion of time to

exhaustion. These findings suggest that there is a strong link between the ventilatory and

perceived exertion responses to cycling following eccentric exercise.

8.1.4 Time to exhaustion

The perception of exertion may be considered fundamental to the individual exercise

response when a participant is required to exercise to ‘volitional exhaustion’. Observations

of elevated RPE have previously been associated with reduced time-trial performance in

running and cycling following EIMD (Marcora & Bosio, 2007; Twist & Eston, 2009).

During incremental cycle exercise in study 2 (Chapter 5) and incremental knee extensor

exercise in study 3 (Chapter 6) the observed decrease in time to volitional exhaustion and

the associated reduction in peak work rate values were consistent with observations of

impaired time trial performance brought about by the effects of eccentric exercise.

Similarly, in studies 1 and 4 (Chapters 4 and 7) time to exhaustion was reduced following

eccentric exercise, during constant load cycling at an intensity of 70%Δ. The precise nature

of the accelerated fatigue development experienced with EIMD is poorly understood. Both

central and peripheral fatigue factors have been implicated in the decreased endurance

capacity observed following EIMD.

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8.1.5 The metabolic response to dynamic exercise with EIMD

Central fatigue factors such as the enhanced production of inflammatory cytokines within

regions of the brain responsible for movement, motivation, perception of effort and pain

have been associated with decreased run time to fatigue (Carmichael et al., 2005).

Similarly, peripheral fatigue factors originating within the damaged muscle tissue such as

changes in metabolic function have been implicated in the decreased endurance capacity

observed following EIMD (Asp et al., 1998)

In study 3 (Chapter 6), we used 31P-magnetic resonance spectroscopy (31P-MRS) to

evaluate changes in muscle metabolism during dynamic incremental knee extensor exercise

following eccentric exercise. The reduction in time to exhaustion following EIMD was not

associated with an accelerated depletion of [PCr]. Time to exhaustion during the

incremental knee extensor exercise was reduced by 12% following the muscle-damaging

exercise. However the rate of [PCr] depletion was similar in the two experimental

conditions with end exercise [PCr] remaining 13% higher 48 h after the muscle-damaging

exercise. Increases in [Pi] and [Pi]:[PCr] were observed not only at rest but also during

incremental exercise. However, the rate of increase in [Pi] was not altered as a result of the

muscle damage however the premature termination of the test at 48 h resulted in there

being no significant difference in end exercise [Pi] values. It was tempting to speculate that

the limit to exercise tolerance was moderated by [Pi]. However wide inter-subject

variability was observed with the [Pi] values, thus we could not reliably conclude that [Pi]

was, indeed a limiting factor. Rather that end exercise [Pi] may have made an important

contribution to the reduced time to exhaustion experienced following muscle damaging

exercise.

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8.1.6 Oxygen uptake kinetics and muscle oxygenation

While elevated [Pi] resultant to EIMD may contribute to decrements in performance of

dynamic exercise, microvascular dysfunction observed following EIMD (Kano et al., 2005)

may also contribute to impaired performance due to disruptions to delivery and distribution

of O2 within the capillary bed of the active muscle. We used Near Infrared Spectroscopy

(NIRS) to assess muscle (haemoglobin + myoglobin) oxygenation and to determine the

dynamic balance between oxygen delivery ( Q˙ O2 ) and oxygen uptake ( V˙ O2 )

following the onset of exercise. In particular, the deoxyhaemoglobin (HHb) concentration

([HHb]) NIRS signal was used to non-invasively estimate O2 extraction in the skeletal

muscle microcirculation.

The findings of study 4 (Chapter 7) demonstrated that EIMD resulted in a slowing of

muscle deoxyhaemoglobin concentration [HHb] kinetics without altering pulmonary V˙ O2

kinetics. The observation that the [HHb] kinetics were over 30% slower 48 h after the

performance of eccentric exercise, indicated that there was an increase in the ratio of

oxygen

delivery

oxygen

uptake

( Q˙ O2 : V˙ O2 )

during

transitions

severe-

intensity

exercise. It was proposed that the elevated Q˙ O2 : V˙ O2

ratio demonstrated by the slower

[HHb] kinetic response (i.e. lower [HHb] at a given V˙ O2 across the exercise transition)

was due to compensatory changes to O2 delivery. In summary, these data indicated that

following EIMD, V˙ O2 kinetics were preserved by compensatory increases in local muscle

blood flow which were able to prevent a measurable slowing of V˙ O2 kinetics.

177

8.2 Limitations

During studies 1 and 4 (Chapters 4 and 7) we employed constant-load, exhaustive exercise

protocols in order to study the human response to dynamic exercise. The use of time or

distance trials such as those employed by Marcora & Bosio (2007) or Twist and Eston

(2009) may provide more ecologically valid models with which to study the effects of

EIMD on the human response to dynamic exercise. However, transitions to constant load

exercise provide the only appropriate platform for modelling of the kinetic response. Thus,

whilst we appreciated the low ecological validity of using exhaustive, constant-load

exercise protocols, we believed we were justified the need to employ our chosen exercise

protocols in order to appropriately examine the human response to dynamic exercise with

and without EIMD.

The relatively small sample sizes used for the investigations that comprise this thesis were

approved as adequate to detect clinically or biologically worthwhile results by the Ethics

Committee of the School of Sport and Health Sciences at the University of Exeter

following appropriate, rigorous power calculations (See Appendix A).

All participants were recreationally active, healthy young men and as such interpretation of

our findings should be restricted to matching populations. Extrapolating findings to other

populations such as highly trained or sedentary groups would be inappropriate. The

protective adaptation to a single bout of eccentric exercise, the ‘repeated bout effect’

(Nosaka & Clarkson, 1995) may last up to 6 months for most symptoms of EIMD (Nosaka

et al., 2001a). Thus the training status of participants may have a profound influence on

their response to dynamic exercise with EIMD.

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8.3 Implications and future directions

The main findings of the studies that comprise this thesis have implications for the

interpretation of human responses to physical exercise following eccentric exercise. An

awareness of potential changes in metabolic, ventilatory, gas exchange and perceived

exertion responses following eccentric exercise may help coaches, exercise scientists and

health and fitness practitioners to make more informed decisions regarding advice given to

their charges. As has been alluded to earlier in this chapter, the use of constant-load,

exhaustive exercise protocols is not ecologically valid. In future, researchers should seek to

investigate changes in the human response following EIMD using, for example, distance or

time trial protocols, such as those used by Marcora and Bosio (2007) and Twist and Eston

(2009). The increased popularity of high intensity exercise training methods including

resistance training and plyometrics may lead to an increased incidence of EIMD in the active

population. Recreationally active individuals should be aware of the potential alterations to

their performance capacity in the days following this type of training. Future research should

also attempt to elucidate differences in the human response to EIMD between individuals of

different training and activity status.

The influence of eccentric, muscle damaging exercise on the human response to dynamic

exercise has only become the focus of scientific investigation in more recent years (see section

2. 7). Whilst this thesis has extended the examination of some of these responses, many

questions remain unanswered. The various human responses to dynamic exercise evaluated

within this thesis; including ventilatory, gas exchange, perceived exertion and metabolic

responses have only been observed 48 h after EIMD. The use of a discrete observational period

was intentional with the time of 48 h chosen to correspond with the period of maximal muscle

179

soreness. However, future research should attempt to evaluate time-course changes in these

and other human responses during the hours and days following eccentric, muscle damaging

exercise in order to elucidate any potential mechanistic links with time-course changes in

known markers of EIMD such as the loss of force generating capacity.

In studies 3 and 4 (chapters 6 and 7) we used animal studies (Carmichael et al., 2005 and

Kano et al., 2005, chapters 6 and 7 respectively) to inform our understanding and provide

the basis of the research question in human subjects. Understandably, we have not been

able to use some of the techniques employed with animal models. Carmichael et al. (2005)

dissected the brains of eccentrically exercised mice to reveal that increases in inflammatory

cytokine concentrations in the cortex and cerebellum were associated with reduced exercise

tolerance. Inspired by the invasive intravital microscopy studies of rat spinotrapezius

muscle by Kano et al. (2005), we used NIRS to facilitate the non-invasive assessment of

muscle oxygenation following EIMD. In study 4 (chapter 7) we observed a reduced [HHb]

signal in the vastus medialis muscle for a given V˙

at exercise onset 48 h during cycling

after squatting exercise. Future research could extend the use of NIRS to facilitate the non-

invasive assessment of muscle oxygenation following EIMD by examining the response to

a range of different damage and exercise protocols.

8.4 Conclusion

In conclusion, the studies that comprise this thesis have demonstrated how the human

response to dynamic exercise may be altered following exercise-induced muscle damage.

The structural and functional changes that characterise EIMD have been demonstrated to

generate additional or altered ventilatory stimuli during exercise such that the ventilatory

180

response to cycle exercise was elevated at exercise intensities above and below the gas

exchange threshold. Furthermore, the augmented ventilatory response observed has been

shown to result in the gas exchange threshold occurring at a lower work rate without a

concomitant change in the lactate threshold. In combination, these findings demonstrate

that the enhanced ventilation experienced following EIMD may be controlled by additional

or altered neurogenic stimuli rather than changes in metabolic factors. Increases in the

ventilatory response were associated with elevated ratings of perceived exertion (RPE) and

a reduction in time to exhaustion. Differences in minute-by-minute ventilation and RPE

values were eliminated when expressed as a percentage of total exercise duration. These

findings indicate that there is a strong link between the ventilatory and perceived exertion

responses to cycling following EIMD. Although we have shown that an increase in blood

[La] is not an obligatory consequence of EIMD, we have demonstrated, using 31P-MRS,

that muscle metabolic response to dynamic exercise is altered following EIMD. The

accelerated fatigue observed following EIMD may be related either to the increased [Pi]

that was observed at rest and throughout incremental exercise, or to other unmeasured

peripheral or central factors. Finally, we showed that peripheral microvascular dysfunction

resultant to EIMD may contribute to impaired performance due to disruptions to delivery

and distribution of O2 within the capillary bed of the active muscle. Using Near Infrared

Spectroscopy (NIRS) we demonstrated that EIMD resulted in a slowing of muscle

deoxyhaemoglobin concentration [HHb] kinetics without altering pulmonary V˙ O2 kinetics.

Findings demonstrated that there was an increase in the ratio of oxygen delivery to oxygen

uptake ( Q˙ O2 : V˙ O2 ) following EIMD which was due to compensatory changes to

O2 delivery.

Impact of Exercise-Induced Muscle Damage on Human Performance During Dynamic Exercise

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