2.1. Study Participants
Study participants were invited to take part in this study if they met the inclusion criteria: Male 18 to 55 years of age, not engaged in regular resistance training (less than 3 sessions per week), body mass index (BMI) under 30 (non-obese), healthy and at low-risk status for cardiovascular disease, and willingness to avoid the use of protein and large-dose vitamin/mineral supplements (above 100% of the recommended dietary allowances), herbs, and all medications (in particular, non-steroidal anti-inflammatory drugs or NSAIDs, such as ibuprofen and aspirin) during the week of the project. Participants voluntarily signed the informed consent, and procedures were approved by the university Institutional Review Board (18-0165). Trial Registration: ClinicalTrials.gov, U.S. National Institutes of Health, identifier: NCT03448328.
The Consolidated Standards of Reporting Trials (CONSORT) flow diagram is shown in Figure 1
. After n
= 136 participants were assessed for eligibility, n
= 109 were randomized by A.J.S. to one of three groups (whey protein, pea protein, water), and n
= 92 received and completed the allocated intervention.
2.2. Study Design and Protein Supplementation Protocol
Participants reported to the Human Performance Laboratory (HPL) at the North Carolina Research Campus, Kannapolis, NC, prior to the start of the study for an orientation to the study procedures and body composition testing. Body composition was measured with the Bod Pod body composition analyzer (Life Measurement, Concord, CA, USA). The participants were randomized to one of three parallel groups using a 1:1 allocation (randomization.com): Pea protein, whey protein, or water only. Data for this study were collected during 2018 and 2019. Study participants continued their normal physical activity and food intake schedules during the 5-day study. Physical activity and food intake were not measured before or during the study.
Participants returned to the HPL in an overnight fasted state for day 1 (Mondays) sample collection, physical fitness testing, and the eccentric exercise bout. Participant ratings of the delayed onset of muscle soreness (DOMS) scale (1–10) were recorded prior to blood sampling [37
]. Protein supplements (0.3 g protein/kg body mass) with 237 mL of water or water alone (237 mL) were ingested. Pea protein isolate (85.2% protein) (NUTRALYS®
S85 Plus, Roquette, Lestrem, France) and whey protein isolate (97.4% protein) (biPro, Eden Prairie, MN, USA) were administered under double blind procedures in shaker bottles. Gram amounts for the protein isolates were adjusted for the differences in protein percentages to ensure that exactly 0.3 g of pea or whey protein/kg were ingested. The average dietary protein intake using national data for the age group selected for this study (ages 18 to 55 years) is 103 ± 4.3 g or approximately 1.25 g/kg per day for non-obese males [38
]. Thus, dietary intake for protein combined with the supplement was calculated at the high but safe level of 2.15 g/kg per day, well below the 3.5 g/kg tolerable upper level [39
Physical fitness testing was initiated within 5 min after the supplements were ingested. Participants were tested for bench press lifting performance, vertical jump performance, leg-back strength, and anaerobic power with the 30-s Wingate test. In the bench press to exhaustion, participants laid down supine on the bench, and bench pressed a weighted bar equal to 75% of their body weight as many times as possible at a rate of 30 lifts per minute. In the vertical jump test, participants jumped as high as possible with one hand, and tapped the measuring device (Vertec vertical jump apparatus, Questtek Corp, Northridge, CA, USA). This test was repeated three times, with the best score recorded as the difference between the jump and standing reach heights. Leg/lower back strength was assessed with a dynamometer (Lafayette Instruments, Lafayette, IN, USA). With the legs slightly bent at the knee, participants grasped a bar attached via a chain to the dynamometer with straight arms, and then lifted with maximal effort for several seconds. This test was repeated three times, with the best score recorded. The Lode cycle ergometer (Lode B.V., Groningen, Netherlands) was used for the 30-s Wingate cycling test. The workload was adjusted to the body mass of the subject (0.7 Newton meters per kilogram), and participants cycled at maximal speed for 30 s. The peak and total wattage power output were recorded and adjusted to body mass.
Study participants next engaged in a 90-min eccentric exercise bout (Table 2
) that consisted of 16 different exercises, most with 2 to 3 sets and 30–60 s of rest between sets [40
Immediately following the 90-min eccentric exercise bout, study participants provided a muscle soreness rating and a blood sample, ingested another protein dose (0.3 g/kg) (or water), and then repeated the four physical fitness tests. Study participants refrained from any food or beverage intake (except for water) for one hour after taking the second supplement dose. Participants were supplied with protein supplements or just water in a shaker bottle (0.3 g/kg in one cup of water) and told to consume these just before going to bed that Monday evening.
Participants returned at 7:00 a.m in an overnight fasted state four days in a row (Tuesday through to Friday) after the eccentric exercise bout (Monday), and provided each morning a DOMS rating and blood sample followed by ingestion of protein (0.3 g/kg) with water or water only. Following ingestion, participants repeated the four physical fitness tests. Participants ingested another protein dose (0.3 g/kg in one cup of water) (or water) immediately after the physical fitness tests, and then again just before going to bed each evening (Tuesday, Wednesday, Thursday). The total supplemental protein ingested during the week (13 doses) was 3.9 g/kg.
2.3. Blood Sample Analysis
The primary outcome measures were serum muscle damage biomarkers, and the secondary outcome measure was C-reactive protein (CRP) (inflammation). Blood samples were collected in serum separator tubes, centrifuged, and analyzed (same day analysis) for serum myoglobin, creatine kinase, lactate dehydrogenase (LDH), CRP, and comprehensive diagnostic chemistries by a commercial laboratory (LabCorp, Burlington, NC, USA).
2.4. Statistical Analysis
The study participant number (n = 30 or 31 per group) provided > 80% power to detect a group difference for creatine kinase and myoglobin with effect sizes > 0.70 at alpha 0.05 using two-sample t-tests. The data are expressed as mean±SE and were analyzed using the generalized linear model (GLM), repeated measures ANOVA module in SPSS (IBM SPSS Statistics, Version 24.0, IBM Corp, Armonk, NY, USA). The statistical model utilized the between-subjects approach: 3 (groups) × 6 (time points) repeated measures ANOVA. This paper provides the time effect (collective effect of the eccentric exercise bout), the treatment effect (whether the treatment groups differed, main effect), and the interaction effect (whether the data pattern over time differed between groups). If the interaction effect was significant (p < 0.05), then post-hoc analyses were conducted using Student’s t-tests comparing time point contrasts (A to B, A to C, A to D, A to E, A to F) between groups. An alpha level of p < 0.01 was used after Bonferonni correction for 5 multiple tests. Cohen’s d was calculated as the difference between the means divided by the pooled standard deviation, with effect sizes regarded as small, medium, and large using cutoff magnitudes of 0.20, 0.50, and 0.80, respectively.