Participant characteristics
Prior to initiating this study, the protocol was reviewed and approved by the Auburn University Institutional Review Ethics Committee (protocol #14-328), and was in compliance with the Helsinki Declaration. Apparently healthy males (n = 30) volunteered to take part in this investigation. Subjects gave written consent and completed the Physical Activity Readiness Questionnaire as well as a health history questionnaire to detect potential risk factors that might be aggravated by strenuous physical activity. All participants were resistance-trained, participating in ≥3 days per week of resistance exercise for at least 3 months.
Experimental protocol
Figure 1 provides an outline of the experimental protocol. The protocol is described more in-depth below and the experimental procedures used in the protocol are described thereafter.
Day 1 (T1)
Participants reported to the laboratory following a 4 h fast. In addition, participants were asked to forgo any strenuous activity for at least 48 h prior to arrival in order to minimize any residual markers of muscle damage. To assure adequate hydration status, urine specific gravity (USG) was measured using handheld refractometer (ATAGO 2393, Bellevue, WA, USA). The hydration threshold prior to exercise testing included a USG of 1.020 g•mL-1. For subjects that were dehydrated, 0.5 L of water were provided and USG was re-determined 30 min later. Baseline venous blood samples were then collected into a 5 mL serum separator tube and a 3 mL EDTA tube (BD Vacutainer, Franklin Lakes, NJ, USA) for subsequent analysis of serum and whole blood markers, respectively. Following blood draws, all participants consumed a cereal bar (Kellogg’s Nutri-Grain® bar; 2 g protein, 24 g carbohydrates, 3 g fat, 120 kcal) in order to standardize pre-testing meals. Participants then had their right mid-thigh shaved and alcohol-swabbed for electromyography (EMG) electrode placement and were seated on System 4 Pro Biodex isokinetic dynamometer (BioDex Medical Inc., New York, USA). Bottom start-position knee flexion was set at 90° and full knee extension was set at 10°. Following a familiarizing practice trial, participants performed a maximal knee extensor isometric contraction for 5 s in order to obtain peak torque and EMG maximal voluntary isometric contraction (MVIC) values. Following a brief recovery period (~1 min), maximal knee extensor isokinetic torque with EMG activity was measured across 5 repetitions at 60°/s and 120°/s, with a brief rest period (~1-2 min) between bouts.
A 5–10 min recovery period was allowed following isometric/isokinetic dynamometry. Following this recovery period, participants began a one repetition maximum (1RM) back squat protocol. The first set of back squats occurred with the bar only (20 kg). Participants were allowed a 2–3 min recovery period and then performed 5 repetitions at ~50 % of their putative 1RM. Participants were allowed a 2–3 min recovery period and then performed 2 repetitions at ~80 % of their putative 1RM. Thereafter, participants performed 1 rep whereby ~2–5 kg were added until they were unable to achieve a successful lift. To ensure that proper depth on each back squat repetition was accomplished, participants were asked to make contact with a box that was behind them without sitting on it entirely. The box was set at a height where the participant’s femur was perpendicular to the ground during the bottom-eccentric portion of the back squat. Following 1RM back squat testing, participants were scheduled subsequent experimental visits. In addition, they were asked to not perform any strenuous physical activity for at least 48 h prior to their day 2 visit described below.
Day 2 (bout 1)
Approximately one week following day 1 (T1), participants reported to the laboratory 4 h fasted, were checked for hydration status and were given a standardized cereal bar to standardize pre-exercise meals. Participants were then asked to mark on a visual analog scale to indicate their perceived soreness (described below). Participants then performed a warm up protocol of 5 repetitions at 20 %, 40 %, and 60 % of their back squat 1RM with 2–3 min between sets. Following the warm-up protocol, participants performed 10 sets of 5 repetitions at 80 % of their back squat 1RM with a 2–3 min recovery period between each set. If participants were unable to complete the repetitions, a 5 % reduction in resistance weight was employed. Lifting volumes for the bout 1 session were recorded and included dropped weights. Immediately following completion of this protocol, participants were randomly assigned to consume one of two commercial products in 600 mL of tap water and the products were provided to participants by laboratory testers. The composition of each product is described in further detail below:
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1)
BCAAs and CHO (BCAA-CHO) (2 servings of AMINO1, Musclepharm Corp., Denver, CO, USA); per 2 servings: 10 kcal, 3 g L-leucine, 1 g L-isoleucine and 2 g L-valine with 2 g of non-sugar carbohydrates
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2)
42 g of carbohydrates (CHO; Powerade, Atlanta, GA, USA); per serving: 168 kcal, 40 g sugar whereby 57 % of the sugar is fructose, 39 % is glucose, 4 % is sucrose and the remainder of the sugars are trace amounts of lactose and maltose
While these supplements were not standardized to CHO or Calorie content, our intent was to provide a practical comparison between these two products. Alternatively stated, we surmised that (in a real-world setting) participants would either consume a sports drink-like CHO beverage or the experimental BCAA-CHO beverage and, thus, we compared two servings of each supplement regardless of Calorie or CHO content. Finally, we elected to provide participants two servings of BCAA-CHO given that two servings contain a total of 3 g of L-leucine and this amount has been posited to be a ‘threshold’ at which post-exercise muscle protein synthesis is optimized [17]. Of note, the testers and participants were blinded to the supplement conditions whereby BCAA-CHO were packaged in ‘B’ containers, and CHO was packaged in ‘A’ containers by a person in the laboratory who did not interact with persons involved in the study.
Day 3 (bout 2)
Twenty four hours following day 2 (bout 1), participants reported to the laboratory 4 h fasted, were checked for hydration status and blood was drawn/collected into a 5 ml serum tube and 3 ml EDTA tubes. Participants were then asked to mark on a visual analog scale to indicate their perceived soreness. Participants then performed the 10 sets of 5 repetitions at 80 % of their back squat 1RM described during day 2 above. Immediately following this exercise bout, participants were administered the same amount of BCAA-CHO or CHO that they had consumed on day 2 described above.
Day 4 (bout 3)
Twenty four hours following day 3 (bout 2), participants reported to the laboratory 4 h fasted, were checked for hydration status and blood was drawn/collected into a 5 ml serum tube and 3 ml EDTA. Again, participants were asked to mark on a visual analog scale to indicate their perceived soreness, and then perform the 10 sets of 5 repetitions at 80 % of their back squat 1RM described during days 2 and 3 above. Immediately following this exercise bout, participants were administered the same amount of BCAA-CHO or CHO that they had consumed on days 2 and 3 described above.
Day 5 (T2; 48 h following bout 3)
The T2 post-test occurred 48 h following day 4 (bout 3) described above, and the post-testing procedure was identical to the T1 described above. Of note, there was one full day of recovery between bout 3 and T2. While participants did not perform exercise during this recovery day, they were sent home with their respective supplement and were instructed to consume either the BCAA-CHO or CHO in order to further facilitate post-training recovery.
Other notes
Participants were asked to maintain their habitual dietary habits, and to ensure there were no potential between-group nutritional confounders, a four day food log was used to assess caloric and macronutrient intake. Food logs were analyzed for daily macronutrient and Caloric intake values using a free online resource [18]. Moreover, participants reported to the laboratory for testing or resistance training during the same time of day (±2 h).
Experimental procedures
Visual analog scale
An adapted visual analog scale (VAS) was utilized to assess perceived muscular soreness as described previously [19]. Briefly, the scale was a straight line, 100 mm in length, and the participants were asked to “mark on line below indicating how sore you are at this moment”. The researcher explained that the most left aspect indicated no soreness at all, whereas the most right aspect indicates the most soreness that the participant has ever experienced.
EMG procedures
Bipolar Ambu BlueSensor M (Ambu INC, Columbia, MD, USA) surface electrodes (Ag/AgCl) were placed over the muscle belly of the right vastus lateralis (inter-electrode distance 25 mm), parallel to the muscle fibers using techniques described by Basmajian and Deluca [20]. Prior to electrode placement the participants’ mid-thighs were shaved, abraded and cleaned using alcohol swabs. A Noraxon Myosystem 1200 (Noraxon USA INC, Scottsdale, AZ) EMG system was used to obtain measurements of neuromuscular activation during MVIC and isokinetic trials. Surface EMG data were sampled at 1000 Hz. Raw EMG signals were full-wave rectified and filtered using a moving average with a 200 ms window. The MVIC was obtained during the isometric knee torque test. Peak values from MVIC trials were used to normalize peak values obtained during the isokinetic trials. Normalized isokinetic values were subsequently represented as percentage of MVIC.
Whole blood and serum analyses
On the days of blood collection, all 3 mL EDTA tubes were refrigerated at 4 °C. Following all testing for the day, tubes were transported to the CLIA-certified Auburn University Medical Clinic, and complete blood count (CBC) panels were analyzed using Beckman-Coulter DxH 600 Hematology analyzer (Beckman Coulter, Fullerton, CA, USA). Specifically, the following whole blood parameters were determined: total white blood cells (WBCs), neutrophil differentials (absolute counts and percentage of WBCs), lymphocyte differentials (absolute counts and percentage of WBCs), and monocyte differentials (absolute counts and percentage of WBCs).
On the days of blood collection, serum was also obtained from 5 ml serum collection tubes through centrifugation at 3500 x g for 5 min at room temperature. Serum aliquots were then placed in 1.7 ml microcentrifuge tubes and stored at -20 °C until batch-processing for serum myoglobin. A human ELISA for myoglobin was used to determine serum concentrations (Abcam, Cambridge, MA, USA). Of note, it has been shown that increases in serum myoglobin concentration is a valid marker of muscle-damage as well as being more sensitive and less variable than creatine kinase [21].
Statistics
Unless otherwise stated, all variables are presented in figures and tables as means ± standard error values. Unless stated below, an alpha (α) level of p ≤ 0.05 was used to detect between- or within-group differences, and all statistics were performed using SPSS v22.0 (Chicago, IL, USA).
Independent t-tests were performed for pre-study training age, height, weight, BMI, and average daily dietary intakes between groups.
T1 and T2 1RM squat, isometric knee extensor torque, isokinetic knee extensor torque, and isokinetic knee extensor EMG activity were analyzed using 2x2 (group*time) mixed factorial ANOVAs. If a significant time α-value was observed for a dependent variable, subsequent paired samples t-tests were performed within each group. Moreover, given that only one comparison was performed within each group for each dependent variable (i.e., T1 vs. T2), raw p-values were used and Bonferroni adjustments were not applied. If a significant group*time α-value was observed for a dependent variable, subsequent paired sample t-tests were performed as described above and independent sample t-tests were also performed to locate specific differences between groups, respectively. Again, given that only one comparison was performed within or between each group for each dependent variable (i.e., T1 vs. T2), raw p-values were used and Bonferroni adjustments were not applied.
Total lifting volume was analyzed using 2x3 (group*time) mixed factorial ANOVAs and VAS data, and serum myoglobin concentrations and gross immunological variables were analyzed using 2x4 (group*time) mixed factorial ANOVAs. If a significant time α-value was observed for a dependent variable, subsequent pairwise comparisons with Bonferroni adjustments were applied within each group. If a significant group*time α-value was observed for a dependent variable, independent t-tests at each time point with manual Bonferroni adjustments were performed at each time point; thus, for the latter independent samples t-tests regarding VAS and serum data, p < 0.0125 was considered to be significant given that four comparisons were being made and manual Bonferroni adjustments were applied to these data.
It should be finally noted that, for all dependent variables analyzed with 2x2/3/4 mixed factorial ANOVAs, Mauchly’s tests of sphericity were performed to assure that the variances of all groups are equal and that the data were normally distributed. In the event that sphericity was not met, the Huynh-Feldt correction was applied to hypothesis testing.