Nutrition Analysis 2

Experimental Gerontology 83 (2016) 112–119

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Experimental Gerontology

journal homepage: www.elsevier.com/locate/expgero

Effect of creatine supplementation and drop-set resistance training in untrained aging adults

Sarah Johannsmeyer a, Darren G. Candow a,⁎, C. Markus Brahms a, Deborah Michel b, Gordon A. Zello b a Faculty of Kinesiology and Health Studies, University of Regina, Canada b College of Pharmacy and Nutrition, University of Saskatchewan, Canada

⁎ Corresponding author at: Faculty of Kinesiology & Hea 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada.

E-mail address: [email protected] (D.G. Can

http://dx.doi.org/10.1016/j.exger.2016.08.005 0531-5565/© 2016 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history: Received 15 June 2016 Received in revised form 4 August 2016 Accepted 10 August 2016 Available online 11 August 2016

Section Editor: Christiaan Leeuwenburgh

Objective: To investigate the effects of creatine supplementation and drop-set resistance training in untrained aging adults. Participants were randomized to one of two groups: Creatine (CR: n = 14, 7 females, 7 males; 58.0 ± 3.0 yrs, 0.1 g/kg/day of creatine + 0.1 g/kg/day of maltodextrin) or Placebo (PLA: n = 17, 7 females, 10 males; age: 57.6 ± 5.0 yrs, 0.2 g/kg/day of maltodextrin) during 12 weeks of drop-set resistance training (3 days/week; 2 sets of leg press, chest press, hack squat and lat pull-down exercises performed to muscle fatigue at 80% baseline 1-repetition maximum [1-RM] immediately followed by repetitions to muscle fatigue at 30% baseline 1-RM). Methods: Prior to and following training and supplementation, assessments were made for body composition, muscle strength, muscle endurance, tasks of functionality, muscle protein catabolism and diet. Results: Drop-set resistance training improved muscle mass, muscle strength, muscle endurance and tasks of functionality (p b 0.05). The addition of creatine to drop-set resistance training significantly increased body mass (p = 0.002) and muscle mass (p = 0.007) compared to placebo. Males on creatine increased muscle strength (lat pull-down only) to a greater extent than females on creatine (p = 0.005). Creatine enabled males to resistance train at a greater capacity over time compared to males on placebo (p = 0.049) and females on cre- atine (p = 0.012). Males on creatine (p = 0.019) and females on placebo (p = 0.014) decreased 3-MH compared to females on creatine. Conclusions: The addition of creatine to drop-set resistance training augments the gains in muscle mass from re- sistance training alone. Creatine is more effective in untrained aging males compared to untrained aging females.

© 2016 Elsevier Inc. All rights reserved.

Keywords: Sarcopenia Muscle mass Strength Endurance Muscle protein catabolism

1. Introduction

The age-related loss of muscle mass and muscle performance has a negative effect on physical function (Balagopal et al., 1997), which sub- sequently decreases the ability to perform activities of daily living (Manini and Clark, 2013). Two interventions which may benefit aging individuals are creatine supplementation and resistance training (for reviews see Candow et al., 2014; Devries and Phillips, 2014; Gualano et al., 2016). Creatine has been shown to increase intramuscular total creatine (i.e. free creatine and PCr) in aging adults (Brose et al., 2003). The increase in high energy phosphates could allow one to train with a greater volume of resistance training leading to an increase in muscle mass (Chrusch et al., 2001). Furthermore, creatine may have a direct ef- fect on muscle biology by influencing cellular hydration status (Balsom et al., 1995), satellite cell activity (Olsen et al., 2006), anabolic hormone production (i.e. IGF-1) (Burke et al., 2008; Deldicque et al., 2005), myo- genic regulatory transcription factors (Hespel et al., 2001; Willoughby

lth Studies, University of Regina,

dow).

and Rosene, 2003) and muscle protein kinetics (Candow et al., 2008; Parise et al., 2001; Safdar et al., 2008).

Speculation exists that the age-related loss of muscle mass is partial- ly caused by an attenuated response to resistance training (Breen and Phillips, 2011). Therefore, a greater volume of resistance training may be needed to produce significant muscle accretion and strength gains in aging individuals. One potential strategy which may help increase the volume of resistance training performed and subsequently benefit aging muscle is combining heavy loads with light loads in each working set. Young adults who performed 3 sets of light load resistance training (30% 1-RM to muscle fatigue) experienced similar gains in muscle mass compared to 3 sets of heavy load resistance training (80% 1-RM to mus- cle fatigue) after 10 weeks (Mitchell et al., 2012). However, the 80% 1- RM group had superior muscle strength compared to the 30% 1-RM group. In postmenopausal women, 3 sets of light load training (30% 1- RM to muscle fatigue) for 10 weeks produced significant gains in muscle mass and strength (Weisgarber et al., 2015). Results across studies indi- cate that heavy load (80% baseline 1-RM) and light load (30% baseline 1- RM) resistance training increases muscle size and strength. Since aging is characterized by a loss of muscle mass and muscle performance, it is plausible that a resistance training program which combines heavy

113S. Johannsmeyer et al. / Experimental Gerontology 83 (2016) 112–119

and light loads (80% baseline 1-RM + 30% baseline 1-RM) in the same training set (i.e. drop-set) may benefit aging adults.

Based on the potential of creatine for increasing aging muscle mass, strength (for reviews see Candow et al., 2014; Gualano et al., 2016; Devries and Phillips, 2014) and muscle performance (i.e. number of rep- etitions at a specific percentage of maximal strength) (Rawson and Volek, 2003), the purpose of this study was to compare the effects of creatine supplementation and drop-set resistance training to placebo and drop-set resistance training in untrained healthy aging adults. It was hypothesized that creatine supplementation during drop-set resis- tance training would increase muscle mass, upper and lower body mus- cle strength and endurance, functionality and decrease muscle protein catabolism compared to placebo during drop-set resistance training.

2. Methods

2.1. Experimental design and participants

The study was a double-blind, repeated measures design. In order to minimize differences between groups at baseline, participants were matched according to gender, age, weight, height and baseline physical activity status. After exclusion criteria were applied, participants were randomized using a computer generated schedule on a 1:1 basis to sup- plement with creatine monohydrate (CR) or placebo (PLA) during 12 weeks of drop-set resistance training. A research assistant, who was not involved in any other aspect of the study, was responsible for randomization. The primary dependent variables assessed at baseline and after the intervention included: (1) body composition (muscle mass, fat mass), (2) muscle strength (1-RM for the leg press, chest press, hack squat, and lat pull-down exercise), (3) muscle endurance (leg press and chest press exercises; maximal number of repetitions performed at 80% and 70% baseline 1-RM respectively), (4) tasks of functionality (walking speed, balance, hand grip strength) and (5) uri- nary excretion of 3-methylhistidine (indicator of muscle protein catab- olism). In addition, participants filled out a 3-day food diary during the first and final week of training to determine whether total energy (kcal) and macronutrient intake changed over time.

Forty participants (21 males, 19 females) volunteered to participate in the study. Both males and females were included to increase the im- pact of the study findings to the general population. Females were post- menopausal for at least 1 year (defined as having their last menstrual cycle ≥ 1 year prior to the start of the study). Participants were required to fill out a leisure time exercise questionnaire at the start of the study, which indicated the average number of times they performed strenuous (i.e. heart beats rapidly), moderate (i.e. not exhausting) and mild exer- cise (i.e. minimal effort) per week (Godin and Shephard, 1985). Partic- ipants also filled out a Physical Activity Readiness Questionnaire (PAR- Q+), which assessed their ability to participate in the resistance train- ing program. If a participant indicated a contraindication to exercise, they were required to get medical clearance before starting the study.

Participants were excluded if they had taken medications that affect muscle biology (i.e. corticosteroids) or creatine monohydrate ≤ 12 weeks prior to the start of the study; if they had a his- tory of fragility fractures; diseases that affect muscle biology (i.e. Crohn's Disease), if they suffered from severe osteoarthritis; if they had participated in moderate-vigorous resistance training (≥3 times per week) for 6 weeks prior to the start of the study; if they were vege- tarian; if they were planning to travel during the study period for N2 weeks at a time or if they had pre-existing kidney or liver abnormalities.

Participants were instructed not to change their dietary habits, en- gage in any additional physical activity that was not part of their normal daily routine or consume non-steroidal anti-inflammatory drugs during the study as these interventions can affect muscle protein synthesis (Trappe et al., 2002). The Research Ethics Board at the University of

Regina approved the study and participants were informed of the risks and purposes of the study before their written consent was obtained.

2.2. Creatine supplementation and blinding procedures

Creatine (Creapure® AlzChem Trostberg GmbH, Germany) and pla- cebo (Globe Plus 10 DE Maltodextrin, Univar Canada) were adminis- tered in powder form. The creatine supplementation dosage was 0.1 g/kg/day which has previously been shown to be effective for in- creasing muscle mass in aging adults without resulting in adverse events (Candow et al., 2015, 2008). Creatine was mixed with equal parts maltodextrin (0.1 g/kg/day creatine + 0.1 g/kg/day maltodextrin) and the placebo group received 0.2 g/kg/day maltodextrin so the pow- ders were identical in taste, texture, color and appearance. Contents of the creatine monohydrate powder were verified by testing in an inde- pendent laboratory (The Cary Company, Addison IL, USA; creatine purity N 99.9%). On training days, participants consumed half their sup- plement immediately before and half immediately after each exercise session. Creatine supplementation immediately before and immediate- ly after resistance training sessions has a positive effect on muscle mass (Candow et al., 2014), strength (Candow et al., 2015) and muscle pro- tein catabolism in aging adults (Candow et al., 2014). On non-training days, participants were instructed to ingest one third of their daily sup- plement with each meal (i.e. breakfast, lunch, dinner) as creatine reten- tion has been shown to be higher if co-ingested with insulin-simulating nutrients (i.e., glucose or protein) (Jäger et al., 2011). An individual not involved in any other aspect of the study was responsible for mixing and packaging up the supplements in plastic bags and preparing individual study kits. Each study kit contained the participants' supplement for the duration of the study, detailed supplementation instructions, and measuring spoons. Participants were instructed to mix their supple- ment in water but not caffeinated beverages as this has been shown to decrease the ergogenic effects of creatine (Vandenberghe et al., 1996). A retrospective treatment identification questionnaire was adminis- tered to all participants upon completion of the study in order to assess whether participants thought they were administered creatine, placebo, or unsure about what supplement they consumed.

2.3. Drop-set resistance training program

Prior to the start of supplementation, participants became familiar with the resistance training equipment. The resistance training equip- ment consisted of four machine-based exercises, which included two upper body exercises (chest press, lat pull-down) and two lower body exercises (hack squat, leg press) using Atlantis® (Precision Series) and Equalizer machines in the Fitness and Lifestyle Centre at the University of Regina.

Familiarization 1-RM strength testing was performed for each exer- cise. Following a 5-minute warm-up on a stationary cycle ergometer at a self-selected intensity, participants performed two warm-up sets of each exercise to be tested in the following order: 1 set of 10 repetitions using a load, which was determined by each participant to be comfort- able and 1 set of 5 repetitions using a heavier weight. Two-minutes after the warm-up sets, the load was progressively increased for each subse- quent 1-RM attempt. Participants rested (passively) at least 2 min be- tween 1-RM attempts. All participants reached their 1-RM in 6 sets or less. 5 min of passive rest separated each 1-RM assessment between dif- ferent muscle groups.

The first training session of the familiarization phase (5 sessions) was directly supervised by a researcher, blinded to group allocation. Subsequent familiarization and study training sessions were indirected supervised by staff in the Fitness and Lifestyle Centre, University of Re- gina. During the familiarization phase, participants were properly shown how to use the equipment and perform repetitions to muscle fa- tigue using 80% and 30% of their familiarization 1-RM. During the first two familiarization sessions, participants performed 1 set of drop-set

114 S. Johannsmeyer et al. / Experimental Gerontology 83 (2016) 112–119

resistance training for each exercise in the following order: leg press, chest press, hack squat and lat pull-down. In the subsequent familiariza- tion sessions, participants performed 2 sets of drop-set resistance train- ing for each exercise. An important aspect of the resistance training program was that all sets were performed to muscle fatigue (defined as the inability to perform the concentric phase of a muscle contraction; Candow et al., 2011). Baseline 1-RM strength testing was performed no earlier than 48 h after the last familiarization session. The same re- searcher, blinded to group allocation, performed all assessments. During the study, participants exercised 3 days/week, on non-consecutive days, to reduce the risk of injury. Participants performed 2 sets of drop-set re- sistance training for the leg press, chest press, hack squat and lat pull- down exercises. Each set consisted of performing repetitions to muscle fatigue at 80% baseline 1-RM immediately followed by repetitions to muscle fatigue at 30% baseline 1-RM (Fig. 1). The training load did not change during the 12 weeks of training and supplementation, as the purpose of the study was to overload the upper and lower body muscu- lature by increasing the number of repetitions performed to muscle fa- tigue. Participants filled out training logs so that resistance training volume (weight × sets × repetitions) and progression could be deter- mined. Only complete muscle contractions were used in the training volume calculations. Participants were contacted every second week by email to help ensure exercise and supplementation adherence. Addi- tionally, personal meetings were arranged and supervised training ses- sions were offered throughout the program if required.

2.4. Body composition

Whole-body muscle mass and fat mass were assessed by dual-ener- gy X-ray absorptiometry (DXA) using a Hologic® Discovery™ system. Participants were instructed to refrain from intense physical activity for 24 h and food and drink for 3 h prior to their DXA scan. Before scan- ning, participants were required to remove all objects containing metal (i.e. jewelry, glasses, clothing with buttons, and/or zippers). Nuclear Medicine Technologists performed the scans at baseline and at the end of the study. The coefficients of variation were 0.15% for muscle mass and 0.33% for fat mass.

2.5. Strength and endurance

Leg press, chest press, hack squat and lat pull-down strength and leg press and chest press endurance was assessed using a standard testing protocol. Seat position and settings were recorded for each participant to ensure consistency between pre- and post-testing. Muscle strength and endurance was assessed in the following order: (1) leg press strength, (2) chest press strength, (3) hack squat strength, (4) lat pull- down strength, (5) leg press endurance, (6) chest press endurance. 5 min of passive rest separated each assessment and the time of day and order of tests was identical at the beginning and end of the study.

A horizontal, bilateral leg press machine with backrest placed in a seated position was used to measure 1-RM leg press strength and en- durance. Following a demonstration, participants were positioned in the leg press machine so that a 90° angle at the knees was achieved and feet placed shoulder width part. The starting position was near

Fig. 1. Description of th

full leg extension, with knees slightly bent. Participants were instructed to perform the contraction to a knee flexion of 90°, without putting the load down at the end of the movement, followed by a full extension back to starting position. The coefficient of variation was 0.30%.

The 1-RM test for the hack squat was similar to that of the leg press. The same bilateral, horizontal leg press machine was used but the back- rest was adjusted to a supine position. Following a demonstration, par- ticipants were positioned in the hack squat to a 90° knee and hip angle. Feet were placed shoulder width apart. The starting position was near full leg extension. Participants were instructed to slowly flex their knees to a 90° angle and then push the load back to full leg extension without locking the knees. The coefficient of variation was 0.41%.

For chest press 1-RM and endurance, participants were positioned in a vertical bilateral chest press machine with both feet placed on the floor. Following a demonstration, participants were instructed not to lift their hips off the bench or arch their back during the lift. Participants were positioned in the chest press machine so that the adjacent bars lined up mid-chest level. Participants were instructed to grasp the bars (overhand grip) approximately shoulder width apart and push the load away from the body until full extension without locking the el- bows and then lower the load back to the starting position. The coeffi- cient of variation was 0.46%.

The 1-RM strength test for the lat pull-down was conducted on a lat pull-down cable pulley machine. Following a demonstration, partici- pants were instructed to grasp the bar (overhand grip) slightly wider than shoulder width apart, sit down and then tuck their legs under the thigh-pads for support. Participants pulled the bar down to their chin and then slowly returned the load to the starting position without locking the elbows. Shoulders remained stable and torso slightly arched during the exercise. The coefficient of variation was 0.45%.

2.6. Functionality

Handgrip strength was assessed using a handgrip dynamometer (Jamar® Hydraulic Hand Dynamometer by Sammons Preston Rolyan). The test was conducted in an upright standing position and the arm to be tested was flexed at a 90° angle with the elbow by the side of the body. Grip width was adjusted to the participants' hand size. Partici- pants were instructed to squeeze the dynamometer with maximal iso- metric effort for 3 s. Participants performed one practice trial, followed by two test trials for each hand with 1 min rest between trials. The highest value produced from each hand was recorded. The coefficient of variation was 0.34%.

Dynamic balance was assessed by recording the time it took partic- ipants to perform backward tandem walking (i.e. toe to heel) over a dis- tance of 6 m on a 10 cm-wide board that was raised about 4 cm off the ground. The coefficient of variation was 0.36%. Additionally, the number of errors (i.e. number of times the participant stepped off the walking board) during the test was recorded. Participants performed one prac- tice trial, followed by two test trials, for which the average time and er- rors were recorded. Walking speed was assessed by the time required to walk an 80 m course on an indoor track as fast as possible. The coeffi- cient of variation was 1.26%.

e training protocol.

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2.7. Muscle protein catabolism

For the measurement of 3-methylhistidine (3-MH), an indicator of muscle protein catabolism, urine was collected during the last 24 h of a 72 h meat free diet (not protein free) immediately before and imme- diately after the study. A meat-free diet was implemented because meat consumption increases urinary 3-MH values and may falsely represent an increase in myofibrillar protein turnover (Lukaski et al., 1981). The urine collection procedure was to discard the first urination upon wak- ing in the morning and then collect all urinations for 24 h, including the first urination upon waking the following morning. Participants were instructed to store their specimen in a cold place or fridge during the 24 h urine collection. After completing the 24 h urine collection, partic- ipants recorded the total urine volume (ml) and poured off two aliquots into vials, which were then sealed in a plastic bag. The vials were stored in a freezer at −20°C until analyzed. The concentration of 3-MH was measured using gas chromatography mass spectrometry (GC–MS) (Agilent) (Rathmacher et al., 1992). The daily amount of 3-MH excreted by each participant was determined by multiplying the concentration by the 24 h urine volume. This amount of 3-MH was then expressed rel- ative to lean tissue mass (nmol/kg LTM). The coefficient of variation was 0.50%.

2.8. Diet

Dietary intake was recorded during the first and final week of sup- plementation and resistance training to assess differences in total ener- gy and macronutrient composition between groups. Participants used a 3 day food booklet to record food intake for two weekdays and one weekend day. Participants were instructed to record all food items, in- cluding portion sizes consumed for the three designated days.

2.9. Adverse events

In the case of an adverse event, participants were asked to complete an adverse event form in order to provide details on the type of adverse event, the severity (i.e. mild, moderate, severe, or life threatening), the frequency, and the relationship to the intervention (i.e. not related, un- likely, possible, probable, or definite).

2.10. Sample size estimation and statistical analyses

A priori power analysis (G*Power v. 3.1.5.1) showed that 34 partici- pants were required. This calculation was based on a moderate effect size (Cohen's d = 0.25), an alpha level of 0.05, a β-value of 0.8 for a re- peated measures: within-between interactions, ANOVA approach (Faul et al., 2007).

A 2 (group: CR vs. PLA) × 2 (gender: males vs. females) × 2 (time: pre- vs. post-training) repeated measures analysis of variance (ANOVA) was conducted to determine differences between groups and gender over time for the dependent variables of body composition, muscle strength, muscle endurance, balance, hand grip strength, walk- ing speed, 3-MH, diet and training volume progression. If significant main effects or interactions were found, simple main effects analysis followed using Tukey's post hoc tests or t-tests, file splitting and profile plots. A t-test was used to assess baseline characteristics and average total training volume between groups.

Significance was set at an alpha level of 0.05 and all results are expressed as means ± standard deviation. The magnitude of the differ- ence between significant means was determined by eta squared (η2). This is a measure of the effect size and therefore of the proportion of the total variance that can be explained by the effects of the treatment. A η2 value of 0.15 represents large differences, 0.06 represents medium differences, and 0.01 represents small differences. Statistical analyses were performed using IBM® SPSS® Statistics, v. 21.

3. Results

3.1. Participants and adverse events

Of the forty participants who volunteered for the study, 3 partici- pants (2 male, 1 female) did not meet the inclusion criteria and 1 partic- ipant (male) withdrew because of time constraints. Thirty-six participants started the familiarization phase with 3 participants (fe- males) withdrawing because of knee pain, shoulder problems and time constrains. Therefore, thirty-three participants started the supplementation and resistance training portion of the study. Two participants (1 male, 1 female) then withdrew because of health issues that were unrelated to the study (see Fig. 2 for a summary of recruitment, allocation and analysis). Thirty-one participants completed the study. There were no significant differences between the CR and PLA groups at baseline for age, body weight, height, or physical activity status (Table 1).

Following the 12 weeks of supplementation and training, 5 par- ticipants in the creatine group correctly guessed they were receiving creatine, 6 participants incorrectly guessed and 3 participants did not know. In the PLA group, 6 participants correctly guessed they were receiving placebo, 4 participants incorrectly guessed and 6 par- ticipants did not know. Supplementation compliance, based on participant's entries in their logs, was similar between the CR and PLA group (98% for both groups). Twenty-six participants (CR = 12, PLA = 14) were able to provide urine samples at the beginning and end of the study for determination of 3-MH. Twenty-nine partic- ipants (CR = 14, PLA = 15) were able to provide 3 day food records at the beginning and end of the study.

There were no serious adverse events reported over time. How- ever, as expected, most participants experienced muscle soreness in the first few weeks of training. One participant in the PLA group reported back strain during the familiarization phase and did not perform the hack squat during the 12 week study. Another partici- pant was diagnosed with a torn shoulder muscle at the beginning of the study, which was likely related to a past shoulder injury. Two subjects in the PLA group reported gastrointestinal distress at the beginning of the supplementation. One participant in the CR group reported increased sweating/hot flashes while working out as well as during the day.

3.2. Body composition

There was a significant gender main effect (p = 0.003, η2 = 0.28) and a group × time interaction for body weight (p = 0.002; η2 = 0.31). The CR group experienced a significant increase in body weight over time (pre: 78.3 ± 17.3 kg, post: 80.0 ± 18.3 kg) with no change for the PLA group (pre: 81.8 ± 18.9, post: 81.2 ± 18.4 kg).

There was a significant time main effect (p b 0.001, η2 = 0.54), gen- der main effect (p b 0.001, η2 = 0.66) and a group × time interaction for muscle mass (p = 0.007, η2 = 0.24). The CR group increased muscle mass more than the PLA group (Fig. 3). Both groups experienced a de- crease in fat mass over time (p = 0.002, η2 = 0.30) (CR: pre: 27.2 ± 9.5 kg, post: 25.9 ± 9.3 kg; PLA: pre: 27.8 ± 9.0 kg, post: 26.3 ± 8.8 kg), with no differences between groups or gender.

3.3. Muscle strength and endurance

There was a time main effect (p b 0.001, η2 = 0.78) and gender main effect (p b 0.001, η2 = 0.61) for leg press strength and hack squat strength (time main effect: p b 0.001, η2 = 0.70; gender main effect: p = 0.001, η2 = 0.38), with no differences between groups over time. Similar to leg press and hack squat strength, there was a time main ef- fect (p b 0.001, η2 = 0.76) and gender main effect (p b 0.001, η2 = 0.65) for chest press strength. There was also a gender × time interac- tion (p = 0.005, η2 = 0.27). Both males and females increased chest

Fig. 2. Summary of recruitment, allocation and analyses.

116 S. Johannsmeyer et al. / Experimental Gerontology 83 (2016) 112–119

press strength over time (p b 0.001) but the change was greater in males (pre: 82.3 ± 21.6 kg, post: 100.3 ± 26.9 kg) compared to females (pre: 37.7 ± 10.2 kg, post: 46.7 ± 11.0 kg).

Regarding lat pull-down strength, there was a time main effect (p b 0.001, η2 = 0.82), gender main effect (p b 0.001, η2 = 0.82), gender × time interaction (p = 0.005, η2 = 0.28) and a

Table 1 Subject characteristics at baseline for the CR group and PLA group.

Group Age Mass Height Training status

(years) (kg) (cm) Mild/moderate/strenuous exercise (times/week)

CR (n = 14)

58.0 ± 3.0 78.3 ± 17.3 168.2 ± 9.1 3.6 ± 3.1/1.9 ± 2.2/2.3 ± 1.3

PLA (n = 17)

57.6 ± 5.0 81.8 ± 18.9 171.7 ± 12.6 4.0 ± 5.7/3.1 ± 2.8/2.1 ± 2.1

Values are means ± standard deviation.

group × gender × time interaction (p = 0.049, η2 = 0.15). Both males and females increased lat pull-down strength over time (p b 0.001) but the change was greater in males (pre: 80.4 ± 12.5 kg, post: 93.0 ± 15.1 kg) compared to females (pre: 38.8 ± 7.7 kg, post: 45.7 ± 8.7 kg). Splitting the file by group showed that males on creatine increased lat pull-down strength more (pre: 81.8 ± 12.8 kg, post: 98.0 ± 17.2 kg, p = 0.005) than females on creatine (pre: 39.3 ± 5.9 kg, post: 45.8 ± 7.2 kg) with no differences between males and fe- males on placebo (Males: pre: 79.2 ± 12.9 kg, post: 88.6 ± 12.5 kg; Fe- males: pre: 38.2 ± 9.7 kg, post: 45.6 ± 10.6 kg). Splitting the file by gender showed that there were no significant differences between males or females on creatine or placebo. There was a trend (p = 0.053) for males in the CR group to have greater lat pull-down strength (pre: 81.8 ± 12.8 kg, post: 98.0 ± 17.2 kg) compared to males on place- bo (pre: 79.2 ± 12.9 kg, post: 88.6 ± 12.5 kg).

There was a time main effect for leg press (p b 0.001, η2 = 0.77), and chest press endurance (p b 0.001, η2 = 0.72), with no differences be- tween groups or gender (see Table 2).

Fig. 3. Lean tissue mass before and after 12 weeks of resistance training and supplementation for the CR and PLA groups. Values are mean ± standard deviation. *Significantly different after training (p b 0.05). **Indicates that the CR group increased lean tissue mass more than the PLA group (p = 0.007).

117S. Johannsmeyer et al. / Experimental Gerontology 83 (2016) 112–119

3.4. Tests of functionality

There was a time main effect for the number of errors (p = 0.042, η2 = 0.15) and time taken to finish the balance board test (p b 0.001, η2 = 0.70). In addition, there was a group × time interaction for the time taken to finish the balance board test (p = 0.032, η2 = 0.16). Both groups improved performance over time but the magnitude of change was greater in the placebo group (pre: 32.8 ± 12.7 s, post: 22.7 ± 10.9 s) compared to the creatine group (pre: 28.3 ± 9.0 s, post: 22.8 ± 9.0 s). There was a time main effect (p = 0.013, η2 = 0.21) and gender main effect (p b 0.001, η2 = 0.71) for hand-grip strength and walking time (time main effect: p b 0.001, η2 = 0.42; gen- der main effect: p = 0.039, η2 = 0.15), with no differences between groups over time (Table 3).

3.5. Urinary 3-methylhistidine

There was a gender × time interaction (p = 0.010, η2 = 0.26) and a group × gender × time interaction (p = 0.017, η2 = 0.23) for 3-MH. Post hoc analysis showed that males experienced a significant decrease in 3-MH over time compared to an increase for females (Fig. 4). Splitting the file by gender showed that females on creatine increased 3-MH ex- cretion over time, whereas females on placebo decreased 3-MH (p =

Table 2 Muscle strength (1-RM) for the leg press, hack squat, chest press and lat pull down exercise and press and 70% baseline 1-RM for chest press) before and after 12 weeks of supplementation an

1-RM strength (kg) CR group

Exercise Pre Post % p-V

Leg press 117.5 ± 37.0 145.2 ± 37.0 23.6 b0. Hack squat 100.0 ± 37.3 121.6 ± 46.1 21.6 b0. Chest press 62.0 ± 27.4 77.3 ± 32.8 24.7 b0. Lat pull-down 60.6 ± 24.0 71.9 ± 29.9 18.6 b0.

Endurance (number of repetitions)

CR group

Exercise Pre Post % p-Val

Leg press 10.9 ± 4.1 24.2 ± 9.1 122.0 b0.00 Chest press 11.1 ± 2.3 22.6 ± 7.4 103.6 b0.00

Values are mean ± standard deviation. % = percent change over time.

0.014). There were no differences between males on creatine or place- bo. Splitting the file by group showed that males on creatine decreased 3-MH compared to an increase for females on creatine (p = 0.019) with no differences between males and females in the placebo group.

3.6. Training volume and progression

Average total training volume per session (across 4 exercises) was similar between the CR group (12,961.5 ± 6656.9 kg) and the PLA group (13,426.5 ± 4624.6 kg). There were no differences in average training volume between groups for the leg press (CR: 5007.5 ± 3015.3 kg, PLA: 5084.9 ± 2636.5 kg), chest press (CR: 2046.6 ± 972.7 kg, PLA: 2219.9 ± 810.2 kg), hack squat (CR: 3556.1 ± 1835.8 kg, PLA: 3838.5 ± 1700.3 kg) or lat-pull down exercise (CR: 2351.5 ± 1010.4 kg, PLA: 2283.1 ± 712.4 kg).

Regarding the change in training volume from week 1 (average of the first three training sessions) to week 12 (average of the last three training sessions), there was a significant time main effect (p b 0.001, η2 = 0.77), gender main effect (p = 0.002, η2 = 0.33), gender × time interaction (p = 0.005, η2 = 0.27) and group × gender × time interac- tion (p = 0.043, η2 = 0.15). The change in training volume from week 1 to week 12 was greater in males compared to females. Splitting the file by gender showed that males on creatine progressed at a faster rate compared to males on placebo (p = 0.049). There were no differences in training volume progression between females on creatine or placebo. Splitting the file by group showed that males on creatine progressed with a greater volume of training compared to females on creatine (p = 0.012) with no differences between males and females on placebo. Compared to week 1 (average of the first three training sessions), the creatine group performed (on average) 61 more repetitions at 80% base- line 1-RM (CR-males: 71, CR-females: 50) and 79 more repetitions (CR- males: 107, CR-females: 50) at 30% baseline 1-RM after 12 weeks of training (average of last three training sessions). Compared to week 1 (average of first three training sessions), the placebo group performed (on average) 40 more repetitions at 80% baseline 1-RM (PLA-males: 37, PLA-females: 44) and 72 repetitions at 30% baseline 1-RM (PLA- males: 66, PLA-females: 77) after 12 weeks of training.

3.7. Diet

There was a group × gender × time interaction for carbohydrate in- take (p = 0.022, η2 = 0.19). Splitting the file by gender showed that males in the CR group increased carbohydrate intake over time (pre: 250.1 ± 32.3 g, post: 319.1 ± 105.7 g), whereas males in the PLA group decreased carbohydrate intake (pre: 239.0 ± 121.6 g, post: 207.4 ± 100.2 g). There was no change in fat intake between groups or gender during the study (p = 0.055). There was a gender main effect for protein (p = 0.009, η2 = 0.24) and caloric intake (p = 0.023, η2 = 0.19), with males consuming more protein (pre: 103.0 ± 32.0, post:

endurance measurements (repetitions to volitional fatigue with 80% baseline 1-RM for leg d high-low resistance training.

PLA group

alue Pre Post % p-Value

001 126.2 ± 32.9 162.0 ± 40.0 28.4 b0.001 001 102.8 ± 35.1 120.3 ± 36.7 17.0 b0.001 001 61.1 ± 29.8 73.6 ± 36.3 20.5 b0.001 001 60.1 ± 23.9 68.5 ± 24.9 14.0 b0.001

PLA group

ue Pre Post % p-Value

1 10.6 ± 3.4 25.2 ± 9.7 137.7 b0.001 1 15.5 ± 7.0 27.5 ± 15.6 77.4 b0.001

Table 3 Functionality tests (balance test, handgrip strength and walking speed) before and after 12 weeks of supplementation and high-low resistance training.

Functionality tests CR group PLA group

Pre Post % p-Value Pre Post % p-Value

Balance time (sec) 28.3 ± 9.0 22.8 ± 9.0 19.4 0.002 32.8 ± 12.7 22.7 ± 10.9⁎ 30.8 b0.001 Balance errors (steps off the board) 2.3 ± 2.1 1.9 ± 1.8 17.4 0.317 2.1 ± 3.2 1.4 ± 2.6 33.3 0.037 Handgrip strength (kg) 40.1 ± 11.9 41.0 ± 12.8 2.2 0.054 40.0 ± 10.6 41.4 ± 11.1 3.5 0.058 80 m walking time (sec) 38.2 ± 4.6 37.1 ± 4.3 2.9 0.045 36.2 ± 5.7 33.9 ± 6.6 6.4 0.001

Values are mean ± standard deviation. % = percent change over time. ⁎ Indicates PLA had greater improvements in balance time vs. CR (p b 0.032).

118 S. Johannsmeyer et al. / Experimental Gerontology 83 (2016) 112–119

108.8 ± 34.3 g) and having higher total calorie intake (pre: 2186.3 ± 727.2 kcal, post: 2401.5 ± 859.5 kcal) than females (Protein: pre: 83.1 g ± 14.6 g, post: 80.7 ± 13.8 g; Total calories: pre: 1798.1 ± 231.8 kcal, post: 1802.9 ± 367.1 kcal). Total calorie and macronutrients intake between groups over time is shown in Table 4.

4. Discussion

This is the first study to examine the effects of creatine supplemen- tation and drop-set resistance training in aging adults. Results showed that drop-set resistance training increased muscle mass, strength, en- durance, and tasks of functionality and creatine supplementation aug- mented the gains in muscle mass and strength (lat pull-down in males only) and decreased muscle protein catabolism (males only). These results are important because the reduction in muscle mass and strength with aging decreases the ability to perform activities of daily living (Manini and Clark, 2013) and improvements in muscle size may lead to greater functionality over time (Chalé et al., 2013).

Creatine supplementation increased muscle mass (2.8 kg) to a great- er extent than placebo (0.9 kg). These results support the growing body of research showing a beneficial effect from creatine on aging muscle (Brose et al., 2003; Candow et al., 2015, 2014, 2008; Chrusch et al., 2001; Gotshalk et al., 2008; Tarnopolsky et al., 2007). While the mech- anisms explaining the greater increase in muscle mass from creatine re- main to be elucidated, creatine has been shown to influence intracellular osmolarity and cell swelling (Balsom et al., 1995), gene and muscle protein kinetics (Safdar et al., 2008; Willoughby and

Fig. 4. 3-methylhistidine (3-MH) before and after 12 weeks of resistance training and supplementation for the CR and PLA groups. Values are mean ± standard deviation. *Indicates that males experienced a significant decrease in 3-MH over time compared to females (p = 0.010). **Indicates that females on creatine significantly increased 3-MH, whereas females on placebo decreased 3-MH over time (p = 0.014). ***Indicates a significant difference in 3-MH between men and women in the CR group over time (p = 0.019).

Rosene, 2003), satellite cell proliferation and differentiation (Olsen et al., 2006), and anabolic hormone secretion (i.e. IGF-1) (Burke et al., 2008; Deldicque et al., 2005). It is possible that some of the gains in muscle mass observed in the present study were from water retention. Burke et al. (2003) observed an increase in body water retention and muscle mass after eight weeks of creatine supplementation. However, the percentage of water retention to body mass did not differ in subjects supplementing with creatine, suggesting that the increase in water re- tention corresponded to an increase in dry muscle mass.

Males who supplemented with creatine experienced a decrease in urinary excretion of 3-methylhistidine, an indicator of muscle protein catabolism, which supports our previous findings in aging males (Candow et al., 2014, 2008). Furthermore, Parise et al. (2001) showed that creatine decreased protein catabolism in males, not females. Re- sults across studies indicate that creatine's effect on muscle protein ca- tabolism may be gender specific. The anti-catabolic effects of creatine may have enabled males to resistance train at a greater capacity over time. The lack of response from females on creatine could involve intra- muscular creatine metabolism (Dalbo et al., 2009). Females may have higher resting intramuscular creatine levels than males (Forsberg et al., 1991) and therefore may not respond as well to creatine supplemen- tation (Tarnopolsky, 2000).

Creatine supplementation augmented the gains in muscle strength (lat-pull down) in males only. This small beneficial effect could be due to exogenous creatine increasing high-energy phosphate metabolism (Brose et al., 2003; Smith et al., 1998) or calcium reuptake into the sar- coplasmic reticulum (Bazzucchi et al., 2009). Creatine had no greater ef- fect on other indices of muscle strength or tasks of functionality which could be related to the complexity of the exercises used in the resistance training program and the type, frequency and duration of the training program.

Drop-set resistance training improved muscle mass, muscle perfor- mance and functionality in untrained aging adults. These results sup- port the growing body of evidence showing that heavy and light loads, performed to muscle fatigue, are beneficial (Burd et al., 2010; Mitchell et al., 2012; Weisgarber et al., 2015). For example, in postmenopausal women, significant improvements in muscle size (i.e. elbow flexors and extensors) and strength (i.e. biceps curl, leg extension, triceps ex- tension) were observed after 10 weeks of light load (30% baseline 1- RM to muscle fatigue) resistance training (Weisgarber et al., 2015). In young adults who performed unilateral leg extensions at 30% 1-RM to muscle fatigue and 90% 1-RM to muscle fatigue, significant increases in the rates of muscle protein synthesis were observed (Burd et al., 2010). Additional work from the same laboratory showed that 3 sets of light load resistance training (30% 1-RM to muscle fatigue) produced similar gains in muscle mass compared to 3 sets of heavy load training (80% 1-RM to muscle fatigue) in a group of young, healthy men after 10 weeks training (Mitchell et al., 2012). Unfortunately, no comparisons can be made between drop-set resistance training (80% 1-RM immedi- ately followed by 30% 1-RM) and heavy load (80% 1-RM) or light load (30% 1-RM) resistance training which limits our ability to conclude that drop-set resistance training is superior to heavy or light load train- ing in aging adults. Progressive overload was achieved by increasing the number of repetitions, and consequently the training volume, rather

Table 4 Total calorie (kcal/day) and macronutrient (g/day) content of the CR and PLA group for 3 days during the first and final week of supplementation and resistance training.

Dietary intake CR group (n = 14) PLA group (n = 15)

Week 1 Week 12 % p-Value Week 1 Week 12 % p-Value

Total calories (kcal/day) 2097.5 ± 329.5 2301.1 ± 724.7 9.7 0.183 1906.9 ± 732.8 1936.5 ± 702.0 1.6 0.741 Carbohydrates (g/day) 235.1 ± 43.4 264.9 ± 102.3 12.7 0.213 227.7 ± 90.0 211.5 ± 81.5 −7.1 0.168 Fat (g/day) 80.4 ± 19.6 90.4 ± 34.6 12.4 0.199 66.5 ± 32.8 74.0 ± 36.9 11.3 0.156 Protein (g/day) 102.2 ± 21.4 100.4 ± 25.0 −1.8 0.794 85.1 ± 29.2 90.4 ± 33.7 6.2 0.218

Data is based on the average for one day from 3-day food records. Values are means ± standard deviation. % = percent change over time.

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than increasing the training load itself. The training load was based on 80% and 30% baseline 1-RM strength. Subsequently, the training inten- sities would have decreased over time as the participants increased muscle strength.

In summary, the addition of creatine to drop-set resistance training leads to superior gains in muscle mass with males on creatine experiencing additional benefits. Future research should compare crea- tine supplementation and drop-set resistance training to creatine sup- plementation and heavy load and light load resistance training in aging adults. Furthermore, long-term training studies examining the ef- fects of creatine and drop-set resistance training on properties of aging bone are needed.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

Creapure was provided by AlzChem Trostberg GmbH, Germany.

References

Balagopal, P., Rooyackers, O.E., Adey, D.B., Ades, P.A., Nair, K.S., 1997. Effects of aging on in vivo synthesis of skeletal muscle myosin heavy-chain and sarcoplasmic protein in humans. Am. J. Phys. 273, E790–E800.

Balsom, P.D., Söderlund, K., Sjödin, B., Ekblom, B., 1995. Skeletal muscle metabolism dur- ing short duration high-intensity exercise: influence of creatine supplementation. Acta Physiol. Scand. 154, 303–310.

Bazzucchi, I., Felici, F., Sacchetti, M., 2009. Effect of short-term creatine supplementation on neuromuscular function. Med. Sci. Sports Exerc. 41, 1934–1941.

Breen, L., Phillips, S.M., 2011. Skeletal muscle protein metabolism in the elderly: interven- tions to counteract the anabolic resistance of ageing. Nutr. Metab. (Lond) 8, 68.

Brose, A., Parise, G., Tarnopolsky, M.A., 2003. Creatine supplementation enhances isomet- ric strength and body composition improvements following strength exercise train- ing in older adults. J. Gerontol. Biol. Sci. Med. Sci. 58, 11–19.

Burd, N.A., West, D.W.D., Staples, A.W., Atherton, P.J., Baker, J.M., Moore, D.R., Holwerda, A.M., Parise, G., Rennie, M.J., Baker, S.K., Phillips, S.M., 2010. Low-load high volume re- sistance exercise stimulates muscle protein synthesis more than high-load low vol- ume resistance exercise in young men. PLoS One 5, e12033.

Burke, D.G., Chilibeck, P.D., Parise, G., Candow, D.G., Mahoney, D., Tarnopolsky, M.A., 2003. Effect of creatine and weight training on muscle creatine and performance in vegetar- ians. Med. Sci. Sports Exerc. 35, 1946–1955.

Burke, D.G., Candow, D.G., Chilibeck, P.D., MacNeil, L.G., Roy, B.D., Tarnopolsky, M.A., Ziegenfuss, T., 2008. Effect of creatine supplementation and resistance-exercise train- ing on muscle insulin-like growth factor in young adults. Int. J. Sport Nutr. Exerc. Metab. 18, 389–398.

Candow, D.G., Little, J.P., Chilibeck, P.D., Abeysekara, S., Zello, G.A., Kazachkov, M., Cornish, S.M., Yu, P.H., 2008. Low-dose creatine combined with protein during resistance training in older men. Med. Sci. Sports Exerc. 40, 1645–1652.

Candow, D.G., Chilibeck, P.D., Abeysekara, S., Zello, G.A., 2011. Short-term heavy resis- tance training eliminates age-related deficits in muscle mass and strength in healthy older males. J. Strength Cond. Res. 25, 326–333.

Candow, D.G., Zello, G.A., Ling, B., Farthing, J.P., Chilibeck, P.D., McLeod, K., Harris, J., Johnson, S., 2014. Comparison of creatine supplementation before versus after super- vised resistance training in healthy older adults. Res. Sports Med. 22, 61–74.

Candow, D.G., Vogt, E., Johannsmeyer, S., Forbes, S.C., Farthing, J.P., 2015. Strategic creatine supplementation and resistance training in healthy older adults. Appl. Physiol. Nutr. Metab. 40, 689–694.

Chalé, A., Cloutier, G.J., Hau, C., Phillips, E.M., Dallal, G.E., Fielding, R.A., 2013. Efficacy of whey protein supplementation on resistance exercise-induced changes in lean mass, muscle strength, and physical function in mobility-limited older adults. J. Gerontol. Biol. Sci. Med. Sci. 68, 682–690.

Chrusch, M.J., Chilibeck, P.D., Chad, K.E., Davison, K.S., Burke, D.G., 2001. Creatine supple- mentation combined with resistance training in older men. Med. Sci. Sports Exerc. 33, 2111–2117.

Dalbo, V.J., Roberts, M.D., Lockwood, C.M., Tucker, P.S., Kreider, R.B., Kerksick, C.M., 2009. The effects of age on skeletal muscle and the phosphocreatine energy system: can creatine supplementation help older adults. Dynamic Med. 8, 6.

Deldicque, L., Louis, M., Theisen, D., Nielens, H., Dehoux, M., Thissen, J.-P., Rennie, M.J., Francaux, M., 2005. Increased IGF mRNA in human skeletal muscle after creatine sup- plementation. Med. Sci. Sports Exerc. 37, 731–736.

Devries, M.C., Phillips, S.M., 2014. Creatine supplementation during resistance training in older adults-a meta-analysis. Med. Sci. Sports Exerc. 46, 1194–1203.

Faul, F., Erdfelder, E., Lang, A.-G., Buchner, A., 2007. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 39, 175–191.

Forsberg, A.M., Nilsson, E., Werneman, J., Bergström, J., Hultman, E., 1991. Muscle compo- sition in relation to age and sex. Clin. Sci. 81, 249–256.

Godin, G., Shephard, R.J., 1985. A simple method to assess exercise behavior in the com- munity. Can. J. Appl. Sport Sci. 10 (3), 141–146.

Gotshalk, L.A., Kraemer, W.J., Mendonca, M., 2008. Creatine supplementation improves muscular performance in older women. Eur. J. Appl. Physiol. 102, 223–231.

Gualano, B., Rawson, E.S., Candow, D.G., Chilibeck, P.D., 2016. Creatine supplementation in the aging population: effects on skeletal muscle, bone and brain. Amino Acids http:// dx.doi.org/10.1007/s00726-016-2239-7.

Hespel, P., Op't Eijnde, B., Van Leemputte, M., Ursø, B., Greenhaff, P.L., Labarque, V., Dymarkowski, S., Van Hecke, P., Richter, E.A., 2001. Oral creatine supplementation fa- cilitates the rehabilitation of disuse atrophy and alters the expression of muscle myo- genic factors in humans. J. Physiol. 536, 625–633.

Jäger, R., Purpura, M., Shao, A., Inoue, T., Kreider, R.B., 2011. Analysis of the efficacy, safety, and regulatory status of novel forms of creatine. Amino Acids 40, 1369–1383.

Lukaski, H.C., Mendez, J., Buskirk, E.R., Cohn, S.H., 1981. Relationship between endogenous 3-methylhistidine excretion and body composition. Am. J. Physiol. Endocrinol. Metab. 240, E302–E307.

Manini, T.M., Clark, B.C., 2013. What we have learned from exercise and lifestyle trials to alleviate mobility impairment in older adults. J. Frail. Aging. 2, 57–59.

Mitchell, C.J., Churchward-Venne, T.A., West, D.W.D., Burd, N.A., Breen, L., Baker, S.K., Phillips, S.M., 2012. Resistance exercise load does not determine training-mediated hypertrophic gains in young men. J. Appl. Physiol. 113, 71–77.

Olsen, S., Aagaard, P., Kadi, F., Tufekovic, G., Verney, J., Olesen, J.L., Suetta, C., Kjaer, M., 2006. Creatine supplementation augments the increase in satellite cell and myonuclei number in human skeletal muscle induced by strength training. J. Physiol. 573, 525–534.

Parise, G., Mihic, S., MacLennan, D., Yarasheski, K.E., Tarnopolsky, M.A., 2001. Effects of acute creatine monohydrate supplementation on leucine kinetics and mixed-muscle protein synthesis. J. Appl. Physiol. 91, 1041–1047.

Rathmacher, J.A., Link, G.A., Flakoll, P.J., Nissen, S.L., 1992. Gas chromatographic mass spectrometric analysis of stable isotopes of 3-methylhistidine in biological fluids: ap- plication to plasma kinetics in vivo. Biol. Mass Spectrom. 21, 560–566.

Rawson, E.S., Volek, J.S., 2003. Effects of creatine supplementation and resistance training on muscle strength and weightlifting performance. J. Strength Cond. Res. 17 (4), 822–831.

Safdar, A., Yardley, N.J., Snow, R., Melov, S., Tarnopolsky, M.A., 2008. Global and targeted gene expression and protein content in skeletal muscle of young men following short-term creatine monohydrate supplementation. Physiol. Gen. 32, 219–228.

Smith, S.A., Montain, S.J., Matott, R.P., Zientara, G.P., Jolesz, F.A., Fielding, R.A., 1998. Crea- tine supplementation and age influence muscle metabolism during exercise. J. Appl. Physiol. 85, 1349–1356.

Tarnopolsky, M.A., 2000. Gender differences in metabolism; nutrition and supplements. J. Sci. Med. Sport 3, 287–298.

Tarnopolsky, M.A., Zimmer, A., Paikin, J., Safdar, A., Aboud, A., Pearce, E., Roy, B., Doherty, T., 2007. Creatine monohydrate and conjugated linoleic acid improve strength and body composition following resistance exercise in older adults. PLoS One 2, e991.

Trappe, T.A., White, F., Lambert, C.P., Cesar, D., Hellerstein, M., Evans, W.J., 2002. Effect of ibuprofen and acetaminophen on postexercise muscle protein synthesis. Am. J. Physiol. Endocrinol. Metab. 282, E551–E556.

Vandenberghe, K., Gillis, N., Van Leemputte, M., Van Hecke, P., Vanstapel, F., Hespel, P., 1996. Caffeine counteracts the ergogenic action of muscle creatine loading. J. Appl. Physiol. 80, 452–457.

Weisgarber, K.D., Candow, D.G., Farthing, J.P., 2015. Whey protein and high-volume resis- tance training in postmenopausal women. J. Nutr. Health Aging 19, 511–517.

Willoughby, D.S., Rosene, J.M., 2003. Effects of oral creatine and resistance training on myogenic regulatory factor expression. Med. Sci. Sports Exerc. 35, 923–929.