Subject Background and Preparation
Thirteen men (mean age: 26.2±5.3 years; height: 184.3±8.2 cm; weight: 92.9±15.6 kg; barbell back squat 1RM: 146.8±30.6 kg) with a minimum of 6 months of prior experience in the barbell back squat completed the study. All subjects provided written informed consent after approval for the study was granted by the Institutional Review Board at Jacksonville University.
The study utilized a randomized, cross-over, placebo controlled design, as illustrated in Figure 1. Subjects initially attended familiarization and preliminary testing. During this visit, subjects were introduced to the facility and walked through the basic procedures, including muscle soreness assessment, the warm-up protocol, and the vertical jump and handgrip dynamometer procedures, followed by a 1-repetition maximum assessment on the barbell back squat.
For muscle soreness, subjects marked a line on a 10cm scale that corresponded to the amount of muscular soreness they were currently perceiving. The warm-up consisted of 5 minutes on a Monark exercise bike (Vansbro, Sweden) keeping a constant speed of 60-80 revolutions per minute. Following the exercise bike, subjects performed a range of dynamic stretching exercises, including bodyweight squats, lunges, lateral lunges, knee hugs, quad pulls and straight leg marches. Each dynamic stretch was performed for 10 repetitions.
For the vertical jump, subjects were instructed to stand on a NeuroCom Balance Manager forceplate (Natus, Pleasanton, CA) with hands on hips. Subjects were instructed to perform 3 consecutive vertical jumps, as high as possible with minimal time between jumps. Jumps were later analyzed for power using the equation described by Sayers et al. (29).
Grip strength of the dominant hand was measured with a grip strength dynamometer (Hydraulic Hand Dynamometer, North Coast Medical Inc., Morgan Hill, USA). The highest score of three attempts was used in the analyses.
For the 1-Repetition Maximum Testing (1RM), subjects performed squats for 8-10 repetitions at ~50% of estimated 1RM followed by another set of 2-5 repetitions at ~85% of 1RM. Subsequently, 4-5 maximal trials were used to determine the individual’s 1RM, each separated by 2 minutes of rest. The highest load lifted for 1 repetition with correct technique was considered their 1RM.
Following the familiarization visit, subjects took the supplement daily for 1 week prior to the acute resistance exercise protocol (AREP). The experimental supplement was a specialized, proprietary broad spectrum Tart Cherry Extract Powder (NordicCherry® ) manufactured by Specnova, LLC. The subjects received a dose of 500mg in capsule form containing 1.3 kcal, 0.3g carbohydrate, 0g fat and 0.008g protein. For the placebo, the subjects received rice flour. The supplement and placebo were prepared by Specnova, LLC.
Acute Resistance Exercise Protocol (AREP)
Subjects arrived at the Exercise Physiology Laboratory following an overnight fast. Subjects were encouraged to drink 2 cups of water the evening prior and 2 cups of water the morning of the visit to ensure adequate hydration. Before, immediately following, 1 hour and 3 hours after the protocol, blood was drawn from an antecubital vein into a serum vacutainer. Blood was spun at 1,500 x g for 10 minutes at room temperature and serum was aliquoted and stored at -80°C until subsequent analyses. The initial blood draw prior to the AREP took place following 10 minutes of seated rest to ensure a true resting blood draw.
Once the baseline blood sample was taken, the subject began the warm up, as described earlier. Following the warm up, the barbell was loaded with a weight corresponding to 75% of their predetermined 1-repetition maximum. The subject then aimed to perform 10 repetitions of the barbell back squat for 6 sets interspersed with 2 minutes of rest. If the subject was unable to complete 10 repetitions for a given set, the subsequent set was then performed with 70% 1RM. This procedure of reducing the load 5% 1RM for each set where the subject fails to complete 10 repetitions was repeated for all sets. Following completion of the AREP, an immediate post-blood was taken. Subsequent blood draws also took place 1hr and 3hr following the protocol.
At 24 and 48 hours after the AREP, a single blood draw was conducted followed by a measurement of handgrip strength and jump power. The blood draw took place following a 10-minute seated rest period. During the 10-minute rest period, muscular soreness was assessed with a 10cm visual analog scale. Handgrip strength and vertical jump power were then assessed with the procedures previously described.
2 Week Washout
Following the first cycle of testing, the subjects underwent a 2 week washout period. During this time no supplement was consumed and the subjects were instructed to resume their normal diet. Following the two week washout, the subjects completed the entire data collection process a second time (7 day loading period, AREP and recovery visits) with the alternate condition (i.e., supplement or placebo).
Serum was analyzed in bulk at the completion of the study. Protein carbonyls (PC) were quantified by an Enzyme-Linked Immunosorbent Assay (ELISA) kit (OxiSelectTM Protein Carbonyl ELISA Kit, Cell Biolabs, Inc., San Diego, CA). Serum protein concentration was determined for each sample using the Bradford Protein Assay (Bio Basic, Amherst, NY) prior to running the ELISA and samples were run in triplicate per manufacturer’s protocol.
Creatine kinase (CK) activity was assessed using a colorimetric assay (Creatine Kinase Activity Assay Kit, Abcam, Cambridge, MA). Serum samples were diluted 1:5 and readings were taken over time for 10 minutes. Sample preparation and analysis were performed per manufacturer’s protocol.
Creatine kinase isoenzyme MB was assayed by ELISA per manufacturer’s protocol (Human CKMB (CKM/CKB) ELISA Kit, Thermo Scientific, Frederick, MD).
All data are presented as means along with standard deviation. Data were confirmed to demonstrate a normal distribution according to Shapiro-Wilk. Data points greater than 2 standard deviations from the mean were removed as outliers. If the baseline value for a subject was not attainable for a certain variable, the subject was removed from analysis for that variable. Missing data was replaced with the mean delta for that time point. A 2 way repeated measures ANOVA (2 conditions: supplement and placebo; 6 time points) was conducted for PC, CK and CKMB. A 2 way repeated measures ANOVA (2 conditions, 3 time points) was conducted for muscle soreness, jump power and handgrip dynamometer measurements. The Mauchley sphericity test was conducted to confirm homogeneity of variance; if this assumption was violated, the Greenhouse-Geisser adjustment was performed If statistical significance was observed, post-hoc analyses were conducted in the form of paired t-tests on the change from baseline values at each time point, with a Bonferroni correction factor to account for alpha inflation. All statistics were run using SPSS Statistical Software, Version 24 (IBM Corporation, Armonk, NY, USA).