Participants
Twenty-five healthy, resistance-trained, male subjects (28 ± 5y; 176.0 ± 6.5 cm; 83.2 ± 12.1 kg) completed this double-blind study. 33 subjects were recruited, and 3 subjects did not complete the study due to scheduling conflicts, 3 were not compliant with protocols, and 2 sustained injuries during the study unrelated to training or supplementation. All subjects were prohibited from using any supplements not provided in the study except for a multivitamin or protein powder food substitute, which they were not permitted to use within 2 h before or after resistance training sessions. Each subject was required to be capable of lifting 1.5x their bodyweight in the squat and deadlift and 1x bodyweight in the bench press. At baseline, the placebo (PLA) group was able to squat 1.71 ± 0.21, bench press 1.45 ± 0.19, and deadlift 2.17 ± 0.25 times their bodyweight, and the treatment (TRT) group was able to squat 1.66 ± 0.24, bench press 1.31 ± 0.20, and deadlift 1.93 ± 0.27 times their bodyweight. Approval for research with human subjects was obtained from the MusclePharm Sports Science Institute IRB (accredited by the United States Department of Health and Human Services), and protocols conformed to the standards set by the latest revision of the Declaration of Helsinki. No members of the IRB were involved in study conception, design, data collection, data analysis, or data interpretation. Subjects provided their written informed consent prior to participation in the study.
Experimental design
Subjects were randomly assigned to either the PLA (n = 11) or TRT (n = 14) groups. They were instructed to consume 1 serving (2 mL) of either PLA or TRT (elevATP®, VDF FutureCeuticals Inc., Momence, IL; 150 mg) 45 min prior to training on training days or at a similar time of day on rest days. The supplement was provided as a liquid with liposomes (QuSomes, BioZone Laboratories Inc., Pittsburg, CA), and the instructed dose was marked on the dropper provided with the vial. PLA consisted of a flavor-matched liquid with identical liposomes added. A third-party, BioZone Laboratories Inc., assembled the TRT and PLA supplements and provided disclosure as to their exact composition to verify subjects received the supplement as described and nothing more. Despite this assumption, the ingredients were not independently verified, and thus, exact contents cannot be confirmed. Supplement vials were weighed to ensure compliance. Subjects were resistance trained under the guidance of a certified strength and conditioning specialist 3 days per week for 8 weeks followed by a 2 week overreach and 2 week taper phase corresponding to weeks 9–10 and 11–12, respectively, in a design identical to that previously described [16]. A eucaloric diet consisting of 50 % calories from carbohydrates, 25 % from protein, and 25 % from fat was prescribed to all subjects at the onset of the study, and diets were tracked weekly via 3-day food logs. Total calories were determined for each individual based on the Mifflin St. Jeor equation adjusted for activity level [17]. Subjects were measured at weeks 0, 4, 8, 10, and 12 for all performance variables. Variables collected consisted of upper and lower body power, upper and lower body maximal strength, maximal and average anaerobic power, upper and lower body strength endurance, irisin, interleukin-6 (IL-6), IL-15, fibroblast growth factor-21 (FGF-21), myonectin, cortisol, C-reactive protein (CRP), and growth differentiation factor-11 (GDF-11). Blood draws were conducted at weeks 0, 4, 8, and 12.
Resistance training program
The resistance training program performed by all subjects has been previously reported [16]. Briefly, weeks 1–8 (standard resistance training phase) consisted of one muscle hypertrophy-oriented workout, one power- oriented workout, and one strength-oriented workout featuring cycle ergometer Wingates following strength training. The back squat, bench press, and deadlift exercises were performed on each day along with several other multi- and single-joint exercises. Participants rested 48–72 h between each training day. During the overreach phase (weeks 9 and 10), participants performed high volume workouts on Monday through Thursday with a strength-oriented workout or performance testing conducted on Friday for weeks 9 and 10, respectively. The taper phase (weeks 11 and 12) consisted of one power day on Mondays then strength and power days on both Wednesdays and Fridays performed at low volume for back squat, bench press, and deadlift only. One serving (35 g) of a whey protein supplement (Combat, MusclePharm Corporation, Denver, CO) providing 25 g of protein, 5 g of carbohydrate, and 1.5 g of fat was provided to all subjects immediately following exercise on all training days.
Measurements
Measurements rotated between upper and lower body exercises to provide localized rest (about 15–20 min), and they were conducted in the order which they are presented herein. Maximal strength was determined using 1-repetition maximum (1RM) tests in the barbell back squat, bench press (BP), and deadlift exercises. Subjects were required to descend such that the anterior hip crease descended below the top of the knee during the squat 1RM test, to make contact with their chest without bouncing or removing their hips from the bench during the BP 1RM test, and they were prohibited from hitching motions in the deadlift. Total strength was calculated as the sum of squat, BP and deadlift 1RMs. All 1RMs were monitored by a Certified Strength and Conditioning Specialist who is also a competitive powerlifter. Lower and upper body power was determined using a linear force transducer (weightlifting analyzer, TENDO Sport Machines, Slovak Republic) during a vertical jump and BP test. The greatest value out of 3 tests for jump height, peak power, and peak velocity were recorded while using a Vertec to measure height. Upper body power was determined via the BP exercise at 30 % 1RM. Subjects performed 3 sets of 3 repetitions under the same rules as the 1RM test. The greatest value for peak power and peak velocity was recorded. Strength endurance was determined using 50 % of each subject’s 1RM and by having them perform repetitions until reaching muscular failure in the squat and bench press exercises. A repetition was subtracted if the participant rested greater than 1 s at the top for squat or at the top or bottom for bench press. Repetitions performed were recorded, and repetitions x load was used to calculate total work performed. Anaerobic power output was determined using a 30s Wingate anaerobic cycle ergometry test (WattBike, Woodway, Waukesha, WI). Each Wingate test consisted of 1 min of light pedaling (50–60 rpm), a 5 s sprint, 2 min of light pedaling, another 5 s sprint, and another 2 min of light pedaling all against no resistance prior to the 30s test. Seat height was recorded during the first visit for each participant and kept constant for every measure. Subjects were provided strong verbal encouragement throughout the test. Peak power, average power, watt:mass, and average speed were recorded. Measurements were conducted at baseline and repeated following weeks 4, 8, 10, and 12. The week 8, 10, and 12 measurements were taken corresponding to the end of the standard resistance training, over reach, and taper phases, respectively. Test-retest separated by 7 days resulted in an intraclass correlation coefficient > 0.965 for all measures.
Serum analysis
Blood draws were performed via venipuncture by a trained phlebotomist. Following a 10-h fast, all subjects submitted a blood sample for analysis in the morning to control for diurnal variations. Blood was drawn from the antecubital vein into dry serum tubes (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ). Upon clotting, blood was centrifuged and serum was collected for analysis. Serum was centrifuged at 5000 g for 5 min to pellet debris. Irisin (Biovision, Milpitas, CA); myonectin (Aviscera Bioscience Santa Clara, CA); GDF-11 (San Diego, CA); cortisol (Spring Valley, CA); FGF-21, IL-6, IL-15, and CRP (R & D Systems, Minneapolis, MN) were measured using quantitative sandwich ELISA kits, following the instructions provided for each kit. Final reactions were measured using a spectrophotometer (Molecular Devices, Sunnyvale, CA) at 450 nm optical density and final concentration of the samples was calculated using SoftMax Pro 5.4 (Molecular Devices, Sunnyvale, CA) via standard curves for reference.
Statistical analyses
Repeated measures ANOVAs were performed to assess group, time, and group by time interactions with a significant p-value considered as ≤0.05. A Fisher LSD post-hoc analysis was used to locate differences. Significant interactions were further analyzed using dependent and independent T-tests for differences between time and group, respectively. Observed power has been included for variables with a significant interaction. Statistica (Version 10, Statsoft, Tulsa, OK) was used for all statistical analyses.