Grass-Fed and Non-Grass-Fed Whey Protein Consumption Do Not Attenuate Exercise-Induced Muscle Damage and Soreness in Resistance-Trained Individuals: A Randomized, Placebo-Controlled Trial

Abstract Eccentric muscle contractions can cause structural damage to muscle cells resulting in temporarily decreased muscle force production and soreness. Prior work indicates pasture-raised dairy products from grass-fed cows have greater anti-inflammatory and antioxidant properties compared to grain-fed counterparts. However, limited research has evaluated the utility of whey protein from pasture-raised, grass-fed cows to enhance recovery compared to whey protein from non-grass-fed cows. Therefore, using a randomized, placebo-controlled design, we compared the effect of whey protein from pasture-raised, grass-fed cows (PRWP) to conventional whey protein (CWP) supplementation on indirect markers of muscle damage in response to eccentric exercise-induced muscle damage (EIMD) in resistance-trained individuals. Thirty-nine subjects (PRWP, n = 14; CWP, n = 12) completed an eccentric squat protocol to induce EIMD with measurements performed at 24, 48, and 72 h of recovery. Dependent variables included: delayed onset muscle soreness (DOMS), urinary titin, maximal isometric voluntary contraction (MIVC), potentiated quadriceps twitch force, countermovement jump (CMJ), and barbell back squat velocity (BBSV). Between-condition comparisons did not reveal any significant differences (p ≤ 0.05) in markers of EIMD via DOMS, urinary titin, MIVC, potentiated quadriceps twitch force, CMJ, or BBSV. In conclusion, neither PRWP nor CWP attenuate indirect markers of muscle damage and soreness following eccentric exercise in resistance-trained individuals.


Introduction
Unaccustomed eccentric exercise may cause exercise-induced muscle damage (EIMD), which can result in structural muscular disruption and increased pro-inflammatory and oxidative stress.The symptoms of EIMD include decreased: muscular force production, range of motion, dampened proprioception, elevated pain sensitivity, and increased muscular swelling; ultimately reducing both functional and exercise capabilities (Howatson and van Someren 2008;Trost et al. 2011;Doma et al. 2018;Owens et al. 2019).EIMD can result in an escalation in blood markers of inflammation, oxidative stress, and damage that can last for up to 4 days (Mickleborough et al. 2015), and symptoms that can last up to 14 days in individuals who engage in unaccustomed eccentric exercise (Owens et al. 2019).
There appears to be no treatment or nutraceutical modality that 100% prevents EIMD and enhances recovery of muscle function and soreness following a bout of eccentric exercise (Howatson and van Someren 2008;Lewis et al. 2012).Since optimal nutrient intake can play an important role in accelerating the recovery process after damaging exercise, nutritional supplementation strategies to mitigate signs and symptoms of EIMD are of interest (Bongiovanni et al. 2020).It is well known that protein intake plays a critical role in the control of muscle protein turnover in response to exercise (Phillips and Van Loon 2011).However, a number of reviews (Pasiakos et al. 2014;Davies et al. 2018) have indicated that there is limited evidence to suggest a correlation between indices of muscle damage and protein supplementation.In contrast, several studies have suggested that various forms of whey protein can attenuate EIMD and enhance the recovery of force in sedentary males (Buckley et al. 2010), cyclists (Eddens et al. 2017), resistance-trained males (West et al. 2017), and physically active females (Brown et al. 2018).Nonetheless, it has been shown that whey protein supplements are not always beneficial for muscle soreness, damage, and strength post muscle-damaging exercise (Eddens et al. 2017;Hilkens et al. 2021).
Perhaps the type of whey protein could be the reason for the lack of conclusive results.Most whey protein supplements on the market are derived from conventional grain-fed cows, though products derived from pasture-raised grass-fed cows have been shown to have higher levels of omega-3 polyunsaturated fatty acids, lower levels of saturated fatty acids, and a higher concentration of several vitamins (e.g.vitamin E) and minerals (e.g.iron) (White et al. 2001;Knowles et al. 2004;Martin et al. 2004;Średnicka-Tober et al. 2016;Alothman et al. 2019;van Vliet et al. 2021).No studies have examined whey protein from pasture-raised, grass-fed cows and EIMD, despite various bioactive proteins, such as lactoferrin, lysozyme, and β-lactoglobulin, being found in elevated concentrations in pasture-raised grass-fed products (Kuczyńska et al. 2012).These bioactive proteins have been shown to elicit opioid, antioxidant, anti-thrombotic, anti-inflammatory, and immunomodulatory properties (Brandelli et al. 2015;Olvera-Rosales et al. 2022;Samtiya et al. 2022), and thus have the potential to mitigate symptoms of EIMD.
Therefore, the main purpose of this study was to determine the comparative effects of whey protein from pasture-raised, grass-fed cows (PRWP) versus conventional whey protein (CWP) supplementation on indirect markers of muscle damage, elicited by barbell back squats in healthy, young, resistance-trained men and women.We hypothesized that PRWP will confer greater protection than CWP supplementation against symptoms of EIMD that develop following eccentric exercise.

Study design
Subjects completed a total of five visits in this double-blind, randomized, iso-caloric placebo-controlled trial at a single university setting (Indiana University -Bloomington).To avoid the robust and persistent (Nosaka et al. 2001) repeated bout effects that occur following eccentric exercise, a parallel design was used.The study design was selected to simulate a real-world application of consuming various whey protein supplements (e.g.PRWP and CWP) and include a placebo, thrice daily in free-living, healthy, resistance-trained subjects, and effects on indirect markers of muscle damage during the recovery period following eccentric exercise.It has been proposed that using EIMD as an inflammatory model allows for the "precise" assessment of the anti-and pro-inflammatory as well as the immunomodulatory potential of nutraceuticals and supplements in humans (Methenitis et al. 2021).The selected protocol to induce muscular damage and the resulting EIMD symptoms has been utilized previously, resulting in "severe" symptoms of EIMD in resistance-trained individuals (Pearcey et al. 2015).Urine was collected for the assessment of muscle damage via titin.Following the urine sample, indirect measures of muscle damage, including delayed onset muscle soreness, range of motion, pain pressure threshold, maximal isometric voluntary contraction, peripheral fatigue, countermovement jump, and barbell back squat velocity were conducted.These measures were performed in the same order during visits 2-5, indicative of pre-EIMD (V2), 24 h post-EIMD (V3), 48 h post-EIMD (V4), and 72 h post-EIMD (V5).All subjects followed the schematic depicted in Figure 1.

Subjects
Healthy males and females (aged 18-40 years) who had resistance-trained for ≥3 months with ≥3 days/week of resistance training, were free from known metabolic, cardiovascular, and musculoskeletal diseases, and were not allergic to dairy, were considered eligible for this study.Additionally, only subjects considered as low risk for cardiovascular disease with no contraindications to exercise as outlined by the American College of Sports Medicine were accepted into this study (Medicine AC of S 2013).The eligibility criterion for resistance training sessions per week was based on the recommended number of resistance training sessions per week according to the American College of Sports Medicine (Westcott 2009).In addition, all male and female subjects were required to produce a barbell back squat with an estimated one repetition maximum of ≥1.5 and ≥1.25 times their body mass for male and female subjects, respectively (obtained on visit 1 (V1)) (Romero-Parra et al. 2020;Elsworthy et al. 2021).
Since it has been shown that eccentric exercise and heavy strenuous resistance training cause similar levels of muscle damage and recovery rates in both sexes (Häkkinen 1993;Sorichter et al. 2001;Hubal and Clarkson 2009;Radaelli et al. 2014;Lee et al. 2017;Bruce et al. 2020;Morawetz et al. 2020), female subjects were included in this study.Furthermore, it has been suggested that it is premature to conclude that short-term fluctuations in female hormones significantly influence acute resistance exercise performance (Colenso-Semple et al. 2023).A single self-reported menstrual cycle questionnaire taken during V1 identified the current contraceptive use of subjects; six (n = 3 in placebo, n = 2 in CWP, and n = 1 in PRWP) used contraceptives while seven (n = 1 in placebo, n = 2 in CWP, and n = 4 in PRWP) were not and had usual menstruation cycles.All female subjects (n = 7) not on birth control were tested during the follicular phase of their menstrual cycle for V2-V5.The recruitment and data collection occurred between January and April 2022.A total of 39 subjects out of the 46 recruited for this study met the inclusion criteria (Figure 2).The baseline characteristics of the enrolled subjects are presented in Table 1.All subjects were advised verbally and in writing of the benefits and risks of the investigation before signing an informed consent document approved by the Indiana University Institutional Review Board for the Protection of Human Subjects (#12559).Furthermore, the study protocol was registered under ClinicalTrials.govidentifier: NCT05100459.All procedures involving human subjects were conducted in accordance with the Declaration of Helsinki.

Familiarization and estimated one repetition maximum back squat testing
During V1, familiarization procedures to minimize the learning effect were performed, involving indirect measures of muscle damage, pain pressure threshold, range of motion, maximal isometric voluntary contraction, peripheral fatigue, and countermovement jump.The estimated one repetition maximum back squat test employing the Berger prediction equation (Berger 1961) was used.The estimated one repetition maximum testing in V1 determined the weight of the eccentric exercise barbell back squat protocol for V2 as well as the barbell back squat velocity weight in V2-V5.Subjects performed a thorough self-selected warm-up consisting of cycling on an ergometer  1.6 ± 0.5 1.5 ± 0.7 1.5 ± 0.5 0.950 Values are mean ± SD. no significant differences were detected between groups for any variable (p > 0.05).f: females.m: males.Bmi: body mass index.ffm: fat free mass.1rm/body mass: estimated one-repetition maximum barbell back squat divided by body mass.prWp: pasture-raised/grass-fed whey protein.CWp: Conventional whey protein.
followed by back squats with an empty 20 kg Olympic barbell (The Rogue Bar 2.0 -Black Zinc, Rogue Fitness, Columbus, OH, USA) before beginning to add weight (Rogue Echo Bumper Plates V2, Rogue Fitness, Columbus, OH, USA) to the barbell in an equipped squat rack with safety spotter arms (SML-2 90" Monster Lite Squat Stand, Rogue Fitness, Columbus, OH, USA).Subjects self-selected a barbell weight of approximately 60-70% of the estimated one repetition maximum and performed the barbell back squat exercise with maximum effort until muscular fatigue.The barbell back squat exercise was performed while maintaining proper form and adequate range of motion, which was defined as femurs parallel to the ground.The Berger equation used the barbell weight and repetition number to produce an estimated one repetition maximum weight (Berger 1961).To ensure back squat movement consistency, subjects chose either unshod or standard athletic shoes.However, no elevated-heel shoes were allowed, and subjects wore the same pair of footwear throughout the study duration.

Experimental control
Subjects were asked to record all foods and beverages consumed for two 24-h periods before supplementation and twice during the supplementation period, for a total of four dietary recall records that they completed on their own time.This method for food and beverage intake was used to ensure that the dietary habits of subjects did not change throughout the duration of the study.Due to the abundant logistical challenges and the desire to perform a more generalizable study to the "free-living" condition, meals were not provided to the subjects by the researchers during the study.Subjects were told to consume their habitual diets throughout the study period.Dietary recalls were collected and analyzed using the Web-based Automated Self-Administered 24-Hour (ASA24), Dietary Assessment Tool, version 2019, developed by the National Cancer Institute, Bethesda, USA (Subar et al. 2012).Subjects completed the ASA24® themselves after receiving training on the recall software from a registered dietitian during V1, but no dietary counseling was provided.Visits 2 through V5 all occurred the same time of the day ± 1 h to account for diurnal variation.Instructions to subjects for 48 h prior to V2 included: abstaining from any anti-inflammatory/pain medication, strenuous physical activity, resistance training, nutraceuticals/vitamins, ergogenic supplements (e.g.beta-alanine), alcohol consumption, and any recovery modality (e.g.massage, compression garments, electrical stimulation, and cold/heat therapy) until study completion (Fernandes et al. 2019;Pavis et al. 2021).Subjects were instructed not to eat or drink caffeine for three hours before V2-V5.Before any measurements of muscle damage took place during V2-V5, subjects completed a health history update, supplementation compliance, sleep and recovery records (Hooper et al. 1995), and a digestibility questionnaire (Qin et al. 2017).Next, hydration state was measured via urine specific gravity using a handheld refractometer (Pen S.G., Atago, Tokyo, Japan), where urine specific gravity <1.020 indicated euhydration.If subjects arrived at the laboratory with urine specific gravity above 1.020, they were provided with 300 ml of water to consume immediately.Non-euhydration occurred in 18.6% (e.g. 29 out of 156 urine specific gravity tests) of the participants.Height was measured using a stadiometer (Seca® 213, Hamburg, Germany), and the Tanita MC-780U instrument (Multi Frequency Segmental Body Composition, Tanita Corp., Tokyo, Japan) was used to measure weight, body fat percentage, and fat-free mass.Lastly, before undertaking dynamic muscular assessments (maximal isometric voluntary contraction, peripheral fatigue, countermovement jump, and barbell back squat velocity), subjects performed a self-selected warm-up and followed this routine throughout the study's duration.

Urinary titin
Urine, collected mid-stream, was obtained for the quantification of urinary titin by a solid phase sandwich enzyme-linked immunoassay (27900, Human Titin N-Fragment ELISA® Kit, Immuno-Biological Laboratories, Inc., Minneapolis, MN, USA).Urinary titin was corrected for creatinine concentration to minimize the influence of changes in urine concentration on urinary titin.Creatinine concentration was determined by a colorimetric assay kit (500701, Cayman Chemical Company, Inc., Ann Arbor, MI, USA).Urinary titin was determined using the following equation: urinary titin = titin n-fragment (nmol/L)/creatinine (mg/dL) × 100 (Tanabe et al. 2021).In brief, urine was collected in a sterile container and then immediately centrifuged or refrigerated (3 °C) until centrifuged.Samples were centrifuged (model X-22R; Beckman, St. Louis, MO, USA) for 10 min at 1000 g, and then the supernatant was collected in cryogenic storage vials (CryoKING®, Biologix, Jinan, Shandong, China) then stored at −80 °C.Samples were later diluted accordingly and analyzed in duplicate using a commercially available enzyme-linked immunoassay kit with detection by spectrophotometry (Powerwave XS™ Spectrophotometer, Bio-Tek Instruments, Winooski, VT, USA).The intra-assay coefficient of variation was 7.415% for titin enzyme-linked immunoassay kits.

Muscle soreness and pain
Delayed-onset muscle soreness was assessed using a visual analog scale and a retrospective pain questionnaire.The visual analog scale assessed current lower limb pain intensity, after subjects completed a single bodyweight squat with a bottom hold of three seconds, subjects drew a line in a 100-mm scale (no pain on the left to the worst possible pain on the right); visual analog scale was quantified by measuring the distance from the left of the scale to the subjects drawn line (Hilkens et al. 2021).For the measurement of muscle soreness over the preceding 24-h period, a 7-point Likert retrospective pain questionnaire for muscle soreness was used to evaluate retrospective perceived pain during daily life activities, with 0 indicating "complete absence of muscle pain" and 6 indicating "severe muscle pain that limits my ability to move."The visual analog scale was completed at V2-V5 and the retrospective pain questionnaire was completed at V3-V5 (Hilkens et al. 2021).

Range of motion
Hamstring flexibility and stiffness were measured using a sit-and-reach box (Lafayette Instrument Company, Lafayette, IN, USA) test using standard ACSM testing procedures (Brown et al. 2018).In brief, with knees fully extended against the box and hands overhead in a diving-like position, subjects were told to gradually reach forward along the measuring board as far as possible, without inducing pain, and to hold that position for two seconds (Medicine AC of S 2013).This final position was recorded to the nearest 0.5 cm and completed once.

Pain pressure threshold
Muscle tenderness, also known as pain pressure threshold, was quantified using a digital algometer (Force One FDIX, Wagner Instruments, Greenwich, CT, USA) on pre-marked sites at two specific points on the quadriceps (rectus femoris and vastus lateralis) and one on the calf (Brown et al. 2018).The sites were midway between the superior aspect of the greater trochanter and tibia head (vastus lateralis), the midway point between the anterior patella and inguinal fold (rectus femoris), and the medial aspect of the calf at a relaxed maximum girth (gastrocnemius).Subjects were instructed to say "now" the moment they felt pain, rather than pressure.Measurements were taken twice at each site and then averaged.

Maximal isometric voluntary contraction
Knee extensor force during voluntary contractions was used to quantify muscle damage and measured with a calibrated load cell (model Z Tension Load Cell; Dillon, Fairmont, MN, USA).The load cell was fixed to a table and connected to a noncompliant cuff (#2793 -Posey Twice-as-Tough Keylock Cuffs, Ankle, Posey Co., Arcadia, CA, USA) attached just superior to the ankle malleoli of the subject's right leg.The height of the load cell was adjusted to each subject to maintain a direct line with applied force.Subjects lay supine on the table with the right knee joint angle set at 90° of flexion and the trunk/thigh angle at 150°.Three submaximal warm-up contractions of 25%, 50%, and 75% of maximal effort occurred first.Following the warmup, three five-second 100% effort maximal isometric voluntary contractions with one-minute rest were performed.Strong verbal encouragement was given throughout all 100% effort maximal isometric voluntary contractions.The best of the three maximal isometric voluntary contractions was used for analysis (West et al. 2017).The noncompliant strap was attached to the load cell for the measurement of force, as well as connected to a custom amplifier (Hector Engineering Co. Inc., Ellettsville, IN, USA) and ultimately sampled at 2000 Hz and analyzed via AcqKnowledge Software v 5.0 (BIOPAC Systems, Goleta, CA, USA).

Peripheral fatigue
With the same ankle cuff, load cell, amplifier, and software described in the maximal isometric voluntary contraction section, peripheral fatigue was assessed via magnetic stimulation (Magstim 200-2; Jali Medical, Newton, MA, USA) of the femoral nerve, which was used to elicit a quadriceps twitch.A series of two twitches were obtained at varying levels of stimulator intensity: 70%, 80%, 90%, 95%, and 100% of maximal stimulator power output, separated by 30 s to determine when supramaximal stimulation was reached.The position of the double 70-mm stimulator coil was placed over the femoral triangle and adjusted to determine the most effective location for each subject.Coil placement was marked on the subject's skin with an indelible marker to insure repeatability of the location and measurement for the following visits.Potentiated quadriceps twitch force was measured two seconds after a three-second maximal isometric voluntary contraction and was repeated six total times separated by 30 s (Fulton et al. 2020).Twitch force amplitude was calculated as the difference between the peak force induced by the supramaximal stimulation and baseline force.Force values from all six repetitions were then averaged and used for analysis.

Countermovement jump
A linear position transducer (GymAware Powertool; Kinetic Performance Technology, Canberra, Australia) interfaced with an iPad (Apple, CA, USA) was used to calculate countermovement jump height.The linear position transducer was magnetically positioned on the ground, with the tethered cable attached to a 20-kg standard Olympic bar (Rogue Fitness, Columbus, OH, USA).Subjects started in a static standing position with the Olympic barbell placed horizontally across their trapezius.Instructions were given to jump using maximal force to achieve a maximal jump height.Three jumps, with an interval of 20 s between jumps, were performed by each subject (Callaghan et al. 2021).The average jump height of the three (in cm) was used for analysis.

Barbell back squat velocity
Following countermovement jump testing, subjects were given three minutes of passive standing rest before completing a barbell back squat testing protocol described previously (Callaghan et al. 2021).Barbell back squat velocity testing, which utilized the Rogue safety rack, barbell, and weights (Rogue Fitness, Columbus, OH, USA), included three repetitions at 40% estimated one repetition maximum as a submaximal warm-up, followed by two minutes of rest and then three maximal effort repetitions at 50% estimated one repetition maximum.The mean velocity was measured using the linear position transducer (GymAware Powertool; Kinetic Performance Technology, Canberra, Australia) per the load velocity profile relationship (Callaghan et al. 2021).At the end of the barbell back squat velocity protocol, ratings of perceived exertion were obtained using a 10-point ratings of perceived exertion scale.

Muscle damaging exercise protocol via eccentric barbell back squats
After baseline measures of muscle damage were concluded in V2, subjects underwent eccentric exercise consisting of 10 sets of 10 repetitions of barbell back squats (Rogue Fitness, Columbus, OH, USA) at 60% of their estimated one repetition maximum's.The eccentric squats were performed to a predetermined depth of femurs parallel to the floor, which was confirmed via the squat safety rack.The tempo, controlled via an interval timer app (TrainCentric, Ventrk LLC, Bellevue, WA, USA) for each repetition was a four-second eccentric contraction, no pause, and a one-second concentric contraction.Subjects rested for two minutes between each barbell back squat set (Pearcey et al. 2015).Subject back squat tempo confirmation and verbal encouragement was performed by the research team during all eccentric barbell back squat sets.Immediately at the end of the fifth and tenth squat set, ratings of perceived exertion were obtained using a 10-point ratings of perceived exertion scale.Further, the heart rate as continuously recorded using a chest strap (Polar H10 heart rate monitor, Polar Electro Inc., Lake Success, NY, USA) throughout the squat protocol, specifically the heart rate at repetition one, five, and 10 of all 10 eccentric barbell back squat sets.

Immediate post-eccentric barbell back squat measurements
Maximal Isometric Voluntary Contraction: To ensure that at least moderate muscle damage occurred due to the eccentric barbell back squats, maximal isometric voluntary contraction was measured, using the same procedure outlined previously.Moderate muscle damage was defined as reductions in force-generating capabilities of ≥20% following EIMD from baseline maximal isometric voluntary contraction measurements (Paulsen et al. 2012).If a 20% or greater reduction in maximal isometric voluntary contraction force did not occur, two further sets of eccentric barbell back squats were performed until this threshold was met.Ten of the 39 subjects required additional barbell back squats to induce sufficient EIMD.

Supplementation
After confirmation of sufficient muscle damage being induced via the maximal isometric voluntary contraction post-eccentric barbell back squats, subjects consumed either a non-protein-containing iso-caloric placebo (maltodextrin), PRWP, or CWP.Subjects consumed the iso-caloric shakes within ~15 min of completion of the eccentric barbell back squats.Subjects consumed their remaining supplement doses in the mid-morning, immediately post study visit (V2-V4), and pre-sleep for the days following the EIMD bout.A supplemental dose was added for a total of 10 doses throughout the intervention.This was dependent on a subject's V2 time.For example, a 7 am V2 time would be three doses a day for V2-V4, which would be nine doses, and then another dose on V5 before the 7 am visit, totaling to 10 doses.This supplementation period lasted for four days throughout visits V2-V5.CWP (100% Whey Protein Powder, unflavored, TGS Nutrition, Las Vegas, NV, USA) and PRWP (Premium Blend Protein, Pasture Raised, Grass Fed, rBST/rBGH and soy free, unflavored, Muscle Feast, Nashport, OH, USA) were provided in 30-gram doses that were weighed using a food scale; each dose provided ~130 calories, 2 g of total fat, 2 g of carbohydrate, and 25 g of protein.The placebo (Maltodextrin, Bulk Supplements, Henderson, NV, USA) was a 35-gram dose, consisting of ~130 Calories, 0 g of fat, 32 g of carbohydrates, and 0 g of protein.Both WPC blends and placebo were mixed with 250 mL water (Hilkens et al. 2021).At the conclusion of visits V2-V4, subjects received their assigned supplementation as well as directions regarding consumption.To ensure sufficient compliance, a supplementation compliance questionnaire was completed at the beginning of visits V3-V5.Further, verbal directions and digital reminders were sent daily via email and text throughout the study period.

Mass spectrometry
Samples of both whey protein supplements were sent to the Indiana University Chemistry Department, where researchers unaffiliated with the current study analyzed the samples for protein content using mass spectrometry.The mass spectrometry data are provided as supplemental data (Supplemental Digital Content 1, Mass Spectrometry Data).

Randomization and blinding
A computer-generated stratified randomization sequence was created using Excel (Microsoft, Redmond, WA, USA) by an independent researcher not associated with this study.The sequence was stratified by gender (male and female) and by moderate and high lifting ability, to form blocks of six subjects, due to the three treatment groups.Male lifting ability was considered high if the estimated one repetition maximum was ≥1.75 times one's body weight (Banyard et al. 2017) and female lifting ability was considered high if the estimated one repetition maximum was ≥1.5 times one's body weight (Romero-Parra et al. 2020).Another individual, not involved with this study, labeled all protein and placebo supplements in blinded containers.Once recruitment, data collection, and data entry were completed and checked, this information was unblinded to the primary researchers.

Statistical analyses
Data were analyzed using IBM SPSS Statistics for Windows, version 28 (IBM Corp., Armonk, NY, USA).Summary statistics of the mean, standard deviation, median, and range were calculated for all variables of interest.Subject characteristics at baseline (e.g.age, height, weight, and body fat percentage) between groups (PLA vs. CWP vs. PRWP) were compared using a one-way analysis of variance.Subject weight and hydration for V2-V5 were assessed for consistency by calculating the intraclass correlation coefficient.The effect of nutraceutical supplementation on each outcome of recovery (e.g.maximal isometric voluntary contraction, countermovement jump, pain pressure threshold) was assessed using linear mixed models with time (pre, 24, 48, and 72 h post-EIMD) and treatment (PLA vs CWP vs PRWP) as fixed factors, including the interaction between them and subject identification as the random effect.Model assumptions were assessed and found to be met through the evaluation of the normality of the residuals.The statistical significance level was set at α ≤ 0.05.Data are expressed as mean + SD and their 95% confidence intervals (CI).
A power analysis, conducted with G*POWER 3.1.9.7 (University of Kiel, University of Dusseldorf, and University of Mannheim, Germany), determined that 30 subjects were needed with a power of 0.80, an effect size (f) of 0.25, and an α = 0.05 to detect within-and between-group differences.The proposed effect size was determined based on a study that compared the effects of conventional whey protein and an iso-caloric placebo on markers of muscle damage in resistance-trained individuals, following strenuous exercise (West et al. 2017).

Subject supplementation compliance and blinding
Subjects, via self-report, consumed 100% of the supplements provided to them.Additionally, the research team observed and documented 100% compliance when participants consumed supplements at the end of V2-V4.The subject blinding questionnaire showed that 18 of the 39 subjects guessed incorrectly as to what supplement they received, and four of the 39 subjects marked "I do not know" regarding what supplement they received.Furthermore, only one of the subjects indicated their correct choice as "very confident" when selecting the supplement that they received.The majority (56.4% of the subjects) was unaware of what supplement they consumed during the study's duration; therefore, subject blinding was considered acceptable.

Physiological effects of exercise-induced muscle damage bout
The eccentric EIMD bout significantly decreased maximal isometric voluntary contraction pre-and immediately post-eccentric barbell back squats (-36.2 ± 11.6%, 95% confidence intervals (CI), −40.0 to −32.4%, p < 0.001), with no difference between groups (p = 0.722).The mean heart rates for all groups at the end of all squat sets were not significantly different between groups (p > 0.05).Furthermore, ratings of perceived exertion at the end of the 5 th set did not differ between groups (p = 0.349).The three groups' mean ratings of perceived exertion of the 10 th set was 9.6 ± 0.7, with a significant difference between groups (p = 0.005).The mean ratings of perceived exertion's for set ten of the EIMD bout for the PRWP group, CWP group, and placebo group were 9.1 ± 0.9, 9.9 ± 0.3, and 9.8 ± 0.4, respectively.

Muscle soreness, range of motion, and pain pressure threshold
The impact of the EIMD bout on delayed onset muscle soreness assessed using a visual analog scale in all groups is presented in Figure 3. Muscle soreness significantly increased 24 h post-EIMD in all groups (33.8 ± 3.1, 95% CI, 25.5 to 42.0, p < 0.05) and remained elevated 48 h post-EIMD compared to baseline.No significance was found for the overall muscle soreness difference between groups (p = 0.515) or a time x group interaction (p = 0.695).All groups returned to baseline levels of muscle soreness at the 72 h visit post-EIMD.
The percent change in range of motion in the PRWP and placebo groups was unaffected throughout the study period (p > 0.05).The CWP group saw a significant increase in the percentage change in range of motion between 24 h and 72 h post-EIMD (p = 0.025, Δ, 9.4 ± 3.2%; 95% CI, 0.8 to 18.1%).Therefore, the CWP group was the only group to exceed baseline levels of range of motion by 2.4 ± 9.1% at 72 h.Overall, there was no significant difference in the percent change in range of motion between groups (p = 0.738) or a time-by-group interaction (p = 0.719).
The pain pressure threshold percent change for the gastrocnemius in the CWP group was unaffected by the EIMD exercise bout (p > 0.05).The PRWP (p = 0.035, Δ, −16.2 ± 5.8%; 95% CI, −31.7 to −0.7%) and placebo (p = 0.014, Δ, −18.6 ± 6.0; 95% CI, −34.6 to −2.5%) groups' pain pressure threshold percent change for the gastrocnemius decreased significantly at 24 h and returned to baseline levels at the 48-h visit.The placebo group was the only group to exceed baseline pain pressure threshold percent change for the gastrocnemius levels by 6.0 ± 35.3% due to a significant increase from the 24-h visit to 72 h post-EIMD (p < 0.001, Δ, 24.6 ± 6.0; 95% CI, 8.5 to 40.6%).Overall, there were no significant differences in the pain pressure threshold percent change for the gastrocnemius between groups (p = 0.865) or time-by-group interaction (p = 0.828).
The pain pressure threshold percent change for the rectus femoris in the placebo group was unaffected by the EIMD exercise bout (p > 0.05).The PRWP and the CWP groups had significant decreases in the pain pressure threshold percent change for the rectus femoris at the 24 h post-EIMD visit of −20.9 ± 6.7% (95% CI, −38.9 to −2.9%, p = 0.014) and −19.7 ± 7.2 (95% CI, −39.1 to −0.2, (p = 0.046), respectively, but these returned to baseline levels at 48 h.Overall, there were no significant differences in pain pressure threshold percent change for the rectus femoris between groups (p = 0.798) or a time-by-group interaction (p = 0.986).
The percent change in the CWP group vastus lateralis pain pressure threshold did not significantly change during the study period (p > 0.05).The PRWP and placebo groups had significant decreases in the pain pressure threshold percent change of the vastus lateralis at 24 h post-EIMD of −20.3 ± 6.2% (95% CI, −37.0 to −3.6%; p = 0.009) and −18.3 ± 6.5% (95% CI, −35.7 to −1.0%, p = 0.033), respectively, but returned to baseline levels at 48 h.The PRWP group pain pressure threshold percent change of the vastus lateralis significantly increased at 24 h and 72 h (p < 0.001; Δ, 26.0 ± 6.2%; 95% CI, 9.3 to 42.7%); this group was the only group to demonstrate levels (5.7 + 38.1%) above baseline scores.In summary, no significant difference in the pain pressure threshold percent change of the vastus lateralis was observed between groups (p = 0.838) or a time-by-group interaction (p = 0.785).

Maximal isometric voluntary contraction
As shown in Figure 5, maximal isometric voluntary contraction of the knee at 24 h decreased significantly by 10.4 ± 3.6% (95% CI, −20.0 to −0.9%) in the placebo (p = 0.024) and 11.2 ± 3.4% (95% CI, −20.5 to −2.0%) in the PRWP group (p = 0.008) compared to the baseline.No significant difference (p > 0.05) in CWP group's maximal isometric voluntary contraction was observed at any time point during the recovery period.The maximal isometric voluntary contraction in the placebo group returned to baseline levels by 48 h following EIMD, while the maximal isometric voluntary contraction in the PRWP group failed to recover to baseline levels (p < 0.05).The maximal isometric voluntary contraction in the PRWP group was −10.1 ± 3.4% (95% CI, −19.3 to −0.9%) below baseline maximal isometric voluntary contraction levels at 72 h (p = 0.024).No significant difference (p = 0.710) in maximal isometric voluntary contraction percent change was observed at any time point between groups.

Peripheral fatigue
The EIMD bout induced −10.6 ± 3.8%; 95% CI, −20.8 to −0.3% (p = 0.039) and −11.0 ± 4.0%; 95% CI, −21.6 to −0.3% (p = 0.039) percent decreases in peripheral potentiated quadriceps twitch force at the 24 h visit in the PRWP and placebo groups, respectively.The CWP group potentiated quadriceps twitch force was not affected by the EIMD bout at the 24-h mark (p = 0.258) and stayed the same relative to baseline throughout the study.No significant difference (p > 0.05) in potentiated quadriceps twitch force percent change was observed at any time point between groups (Figure 6).

Nutrient intake
Table 2 presents the dietary intake of the subjects before and during the experimental period.Energy consumption (p = 0.822), protein consumption in grams (p = 0.794), carbohydrate consumption in grams (p = 0.434), fat consumption in grams (p = 0.793), protein intake relative to body mass (p = 0.724), carbohydrate intake relative to body mass (p = 0.448), and fat intake relative to body mass (p = 0.643) were not different between groups before the supplemental period.
Intake of carbohydrate relative to body mass was different between PRWP and placebo groups (p = 0.049), but not between CWP and placebo (p = 0.051).Excluding the macronutrients provided by the supplements, no significant differences were found with regard to protein (p = 0.653), carbohydrate (p = 0.940), and fat (p = 0.202) intake relative to body mass during the post-EIMD period between groups.

Subject hydration, sleep, stress, and supplement tolerance data
All subjects' physiological markers were stable during V2-V5 via intraclass correlation coefficients for hydration (0.76), total body water (0.99), and body weight (1.0).Subject self-reported sleep, stress, and fatigue levels via questionnaires during V3-V5 showed no differences between groups (p > 0.05).Sleep rating was described as good/average, stress was low/average, and fatigue was documented as low/average for all subjects throughout the study period.All provided supplements were well tolerated via the subject self-reported digestibility questionnaire as evidenced by a mean score of below 1.0 on a 0-10 scale throughout the study (p > 0.05).

Discussion
The present study has shown that supplementing resistance-trained individuals with either PRWP or CWP is similarly futile in attenuating indirect markers of muscle damage, function, and performance.Our findings of EIMD symptoms experienced during recovery are comparable to those of other research groups using the same barbell back squat protocol as used in this study (Pearcey et al. 2015;Fernandes et al. 2019;Scudamore et al. 2021).Authenticating the very hard to maximal effort ratings of perceived exertion data while performing the EIMD bout, all groups experienced an average reduction of 36% in maximal isometric voluntary contraction immediately after the eccentric barbell back squat protocol.Furthermore, significant delayed onset muscle soreness was experienced by all groups of the well-trained individuals 48 h post-EIMD.

Delayed onset muscle soreness, range of motion, pain pressure threshold, and urinary titin
In the present study delayed onset muscle soreness peaked in all groups at 24 h post-EIMD, which agrees with other studies utilizing the same eccentric exercise protocol (Fernandes et al. 2019;Scudamore et al. 2021).Delayed onset muscle soreness was unaffected by both PRWP and CWP, as all groups returned to baseline levels at 72 h following the eccentric exercise bout.Similar data were observed in other studies, which provided subjects with whey protein post-EIMD (Brown et al. 2018;Hilkens et al. 2021).To determine if inadequate protein supplementation was provided in previous studies using 40 (Brown et al. 2018) and 60 grams/day (Hilkens et al. 2021), the present study provided three 25-gram protein servings spaced throughout each day (totaling to 75 grams of protein/day).Together, the evidence from previous studies, and the data in the present study, have shown that regardless of dose, whey protein supplementation does not mitigate delayed onset muscle soreness during the recovery period from eccentric exercise.Strengthening the delayed onset muscle soreness findings, the 24 h retrospective muscle pain questionnaire data had no significant differences between groups throughout the study period.The lack of between groups significance in this measure and among others in this study may be due to the fact the participants were well-trained.Per the delayed onset muscle soreness data, all subjects responded similarly to the EIMD bout which indicates the natural time course of muscle recovery may not be hastened by nutraceuticals alone.Furthermore, the potential for muscle recovery improvement and the ability to detect statistical significance from whey protein supplementation in our well-trained individuals who respond similarly to EIMD is expected to be less likely compared to untrained individuals.Whey protein supplementation has been shown to enhance range of motion compared to an isoenergetic carbohydrate drink following muscle damaging exercise, specifically at 72 h (Brown et al. 2018).However, in the present study no group experienced significant decrements in range of motion following eccentric exercise, and no significant differences were observed between groups at any time post-EIMD.The lack of effect of whey protein supplementation on range of motion following eccentric exercise has been observed previously (Kim et al. 2017).Although subjects went into what is considered full range of motion in the EIMD squat protocol, having future subjects go lower in the squat by touching the posterior chain into the calf musculature may allow range of motion measurement differences to be detectable.Substantial muscle tenderness, as shown by pain pressure threshold decrements, has been documented at 72 h post-eccentric exercise using the same eccentric squat protocol as used in the present study (Pearcey et al. 2015).However, prolonged pain pressure threshold decrements was not observed in the present study.The protein groups did not differ from the placebo group which has been found previously (Brown et al. 2018).Speculatively, the repeated pressure of the algometer head over the course of the four visits (similar to the repeated bout effect) resulted in microtrauma at the pain pressure threshold locations, which may have nullified any differences in pain pressure threshold following the EIMD bout (Sand et al. 1997).
To assess the extent of muscle damage following the barbell back squat, we measured urinary titin concentration.The urinary level of the titin fragment is considered a noninvasive and sensitive biomarker for muscle damage (Kanda et al. 2017;Lee et al. 2021;Inami et al. 2022).Our urinary titin data followed a similar time course and magnitude as reported by Tanabe and colleagues (Tanabe et al. 2021) who analyzed urinary titin levels in collegiate athletes following eccentric-like activity.Compared to baseline levels only the PRWP group had significantly elevated urinary titin levels at the 24-h post-EIMD visit.In contrast, urinary titin levels in the CWP and placebo groups were unaffected throughout the recovery period.Other findings have shown whey protein supplementation does not mitigate the rise of a number of biomarkers related to muscle damage and inflammation post EIMD (Buckley et al. 2010;Cooke et al. 2010;Hilkens et al. 2021), validating the PRWP data.We may have missed the chance for finding statistical significance in this measure as data indicate the elevation in urinary titin concentrations peaking 96 h post-EIMD (Yamaguchi et al. 2020), hence the null results between groups.

Muscle performance measures: maximal isometric voluntary contraction, peripheral fatigue, countermovement jump, and barbell back squat velocity
In the present study the maximal isometric voluntary contraction data showed that the peak force decreased at 24 h in the PRWP and placebo groups during recovery following eccentric exercise whereas the CWP group experienced no maximal isometric voluntary contraction force decrements.Our maximal isometric voluntary contraction data is supported by Buckley and colleagues, using similar dosages of various forms of whey protein, where maximal isometric voluntary contraction is affected differently 24 h following eccentric exercise (Buckley et al. 2010).Other studies support the ability of whey protein supplementation to restore muscle contractility following resistance training (West et al. 2017;Davies et al. 2018).This action is attributed to dietary protein's ability to increase muscle protein synthesis, dampen pathways associated with protein catabolism, and enhance the remodeling of the force-producing myofibrillar protein stores (West et al. 2017;Davies et al. 2018).Therefore, it is unknown why PRWP did not prevent the maximal isometric voluntary contraction reduction at multiple time points post-EIMD, even at the 72 h time point.The study design may have been a factor in this finding as it has been found that when whey protein is compared against an isocaloric placebo regarding maximal isometric voluntary contraction recovery, whey protein supplementation exerts no superior effect (Brown et al. 2018;Hilkens et al. 2021), supporting the null findings of this study.When using a non-energetic placebo against protein supplementation, benefits are found on muscle recovery post exercise (Cockburn et al. 2010;Howatson et al. 2012).Therefore, increasing caloric intake, rather than solely protein consumption, may support the bodies physiological response post-EIMD and may help with the understanding of the results.
The current study showed that CWP supplementation was protective against peripheral fatigue, as assessed by the quadriceps twitch force, specifically at 24 h during the recovery period following eccentric exercise, while the placebo and PRWP groups experienced significant decreases.Supporting our placebo and PRWP findings 24 h post-EIMD, Fernandez et al. (Fernandes et al. 2019) have shown that peripheral fatigue can be induced using a similar eccentric exercise protocol (e.g.squatting exercise).However, the data regarding the impact of whey protein supplementation on peripheral fatigue during recovery from eccentric exercise are sparse in the literature.Since the macronutrient compositions of the two protein supplements is similar, it is unknown why CWP and PRWP groups responded differently at 24 h post-EIMD visit regarding the potentiated twitch force.To make sense of this, the production process for different whey protein supplements, given in equal macronutrient amounts, has been found to have diverse physiologic effects similar to the findings herein in regards to faster muscle recovery after training and enhancing long-term neuromuscular adaptations (Garcia-Vicencio et al. 2018).
Studies have shown that jumping ability (e.g.countermovement jump) is significantly decreased following EIMD (Pearcey et al. 2015;Callaghan et al. 2021).However, in the present study countermovement jump performance at 24 and 48 h during recovery from eccentric exercise was not impacted by PRWP, CWP or placebo supplementation.It has been found that whey protein supplementation can outperform an isocaloric carbohydrate placebo in restoring countermovement jump height just 10 h post-EIMD (West et al. 2017), indicating that this finding may have been missed in the present study because the earliest countermovement jump performance post-EIMD was assessed at 24 h.Unexpectedly, at the 72-h post-EIMD visit the placebo group significantly outperformed both whey protein supplements in countermovement jump performance.Although whey protein supplementation is not detrimental to countermovement jump in this study, additional carbohydrate intake may be superior for countermovement jump performance post-EIMD.The amount of carbohydrates consumed relative to body mass in the placebo group (4.5 g/kg) in the present study is as beneficial as high carbohydrate diets (6.5 g/kg) in optimizing the jump squat performance (Hatfield et al. 2006).Thus, a certain carbohydrate threshold may be necessary because both protein blends averaged ~3.15 g of carbohydrate per kilogram of body mass, which was well below the placebo group's intake of 4.5 g/kg that improved the high-intensity movement performance (e.g.countermovement jump).Future research should establish a relative body mass carbohydrate intake needed to optimize explosive functional movement following EIMD.
Barbell back squat velocity is highly relevant to athletes because this measure can be used to assess training readiness (Callaghan et al. 2021).Eccentric-like exercise activity has been found to significantly reduce barbell back squat velocity for at least 48 h (Callaghan et al. 2021).In the present study both whey protein groups experienced significant barbell back squat velocity decrements, specifically during the visit at 24 h post-EIMD.The PRWP group's barbell back squat velocity returned to baseline levels within 48 h, whereas the CWP group did not fully recover until 72 h post-EIMD.This is the first muscle performance measure where PRWP benefited muscle recovery to a greater degree than CWP.However, the reason behind this outcome is equivocal.The isoenergetic carbohydrate placebo supplement was protective against any barbell back squat velocity reduction post-EIMD, which supports research that has shown higher carbohydrate intake is important in maximizing dynamic and high-intensity movements (Haff et al. 2003;Wax et al. 2010;Hilkens et al. 2021).

Effects of whey protein supplementation on muscle recovery and limitations
The present study does not support protein supplementation to reduce and manage EIMD symptoms (Pasiakos et al. 2014;Eddens et al. 2017;Hilkens et al. 2021).In support of our data, Hilkens et al. (Hilkens et al. 2021) showed that a carbohydrate control outperforms protein supplementation in isokinetic measures of muscle function post-EIMD while not impacting biomarkers of muscle damage, delayed onset muscle soreness, and maximal isometric voluntary contraction (Hilkens et al. 2021).However, exercising individuals should not neglect protein consumption due to its pro-muscle protein synthesis, anti-catabolic, and other crucial health-promoting effects.It is recommended that exercising individuals who want to build muscle tissue consume 1.4-2.0g of protein per kg of body weight each day (Kerksick et al. 2017).In our study, the two protein groups exceeded this protein recommendation while the placebo group followed it: PRWP group (2.6 g/kg), CWP group (2.7 g/kg), and placebo group (1.9 g/kg).Contrary to the present study's research findings, data in the literature find that additional protein intake beyond the recommended levels helps alleviate some symptoms of EIMD in well-trained individuals (Lollo et al. 2014;Brown et al. 2018).
The current placebo group consumed an average of 1.9 g/kg of protein during the post-EIMD period, which fits within the recommended protein intake for building muscle (Kerksick et al. 2017).Because protein is not as abundantly stored in the body as carbohydrates are in the form of glycogen, an apparent ceiling-like effect may be present with protein consumption beyond this study's placebo group intake of 1.9 g/ kg of body mass.Furthermore, 1.9 g/kg is slightly higher than 1.8 g/kg of protein consumption, which is found to maximize muscle protein synthesis (Phillips and Van Loon 2011) and affect lean mass similarly to 4.4 g/kg over eight weeks in resistance-trained individuals (Antonio et al. 2014).Finally, a trial examining markers of muscle damage post-EIMD in resistance-trained individuals found consuming 1.8 g/kg of protein to be comparable to consuming 2.8 g/kg (Roberts et al. 2017).These data and the findings of this study support a potential ceiling effect of protein consumption of 1.9 g/kg of body mass while evaluating muscle recovery and maintenance post-exercise.
Given that lower body exercise decreases glycogen stores and eccentric exercise impairs glycogen resynthesis (Costill et al. 1990), this may partially explain why the isocaloric carbohydrate-based placebo was more effective in the muscle function tests than whey protein supplementation.Muscle damage associated with eccentric exercise has been shown to lower the rate of muscle glycogen storage, specifically at 72 h post-EIMD (Costill et al. 1990), which is where we observed significant differences in the barbell back squat velocity and countermovement jump data between the carbohydrate (placebo) and protein supplementation groups.These physiological findings regarding eccentric exercise and glycogen may help justify this study's findings of carbohydrate supplementation being preferred over protein supplementation to recover muscle function post-EIMD if specific relative protein intakes are achieved (1.9 g/kg).Resistance-trained individuals looking for optimal muscle function recovery should not just focus solely on protein consumption but also on achieving an optimal carbohydrate intake level to fuel muscle recovery and performance.The examination of the macronutrient intake levels of each group revealed that the CWP and PRWP groups had carbohydrate-to-protein intake ratios relative to the body mass of 1.1 and 1.2, respectively.In contrast, the placebo group had a carbohydrate-to-protein intake ratio of nearly 2.4.Importantly, resistance-trained individuals looking to restore muscle function post-EIMD can add maltodextrin to their daily dietary intake to assist the attenuation of EIMD due to the data herein.Although the observed improvements in muscle performance measures with carbohydrate supplementation are modest, it is relevant to well-trained individuals who are always looking for a competitive advantage.Future research should focus on the optimal macronutrient intake ranges and combinations of relative protein and carbohydrate supplementation, rather than absolute protein supplementation amounts, in examining nutraceuticals and EIMD recovery.
The lack of an effect of the EIMD stimulus on urinary titin concentration might have been due to the relatively short duration of the sampling period used, since data shows elevations in urinary titin concentrations peaking 96 h post-EIMD (Yamaguchi et al. 2020).However, the initial development of delayed onset muscle soreness, and its persistence 48 h post-EIMD in all provenly strength-trained subjects, including those who consumed the protein supplements, suggests that some level of muscle damage may have been present in all treatment groups, but this did not manifest into significant changes in urinary markers of muscle damage within the timeframe of the protocol.
The mechanisms underlying the diverging effects in the barbell back squat velocity, peripheral fatigue, and maximal isometric voluntary contraction data via whey protein supplementation remain uncertain.Both whey proteins contain ~22% branch-chain amino acids and ~47% essential amino acids.Therefore, other bioactive proteins found using mass spectrometry between the PRWP and CWP may have played a role in the results.Speculatively, various bioactive peptides such as complement C3, lactadherin, and vitamin-D binding protein in the whey protein drinks, as shown in the mass spectrometry data, could play a physiologic role after a strenuous stimulus.More specifically, data shows complement C3 promoting skeletal muscle regeneration (Zhang et al. 2017) and the therapeutic potential of vitamin-d binding protein (Gomme and Bertolini 2004) and lactadherin (Kamińska et al. 2018).Unfortunately, the experimental design used in the current study cannot provide direct evidence, hence these bioactive peptides may a topic of interest for future work.
There were several notable strengths in this study.The present study carefully attempted to produce a robust and carefully controlled experiment that incorporated a well-strategized supplementation plan.All supplementation regimes were matched for calories to eliminate the calories provided as a possible cofounding variable (Richardson et al. 2012).It may be speculated that increasing caloric intake may enhance muscle function recovery.However, all groups consumed the same number of calories during the post-EIMD period.Furthermore, the calorie intake of this study's subjects during the post-EIMD period is supported by the subject's measured body weight, indicative of an intraclass coefficient of 1.0, suggesting that in the present study, the subjects were in caloric balance.Lastly, the appropriate placebo was utilized herein, maltodextrin, which is extensively used in muscle damage studies examining whey protein supplementation versus placebo (Candow et al. 2006;Babault et al. 2015;Sharp et al. 2015;Hilkens et al. 2021).
This study has several limitations that should be noted.Although subjects reported 100% compliance with consuming the provided supplementation, we observed approximately 30% of the doses being consumed in the laboratory setting.The other 70% were consumed away from the investigators supervision and thus were not verified.Although self-reported dietary data were collected, it is not truly known what exactly was consumed by the subjects throughout the intervention.Finally, the data may not be generalizable to other populations, such as the elderly and sedentary individuals.In conclusion, PRWP or CWP do not attenuate muscle damage and soreness following eccentric exercise in resistance-trained individuals.Dr. Albaro Escalera, PhD, CSCS, earned his doctorate from Indiana University-Bloomington in Exercise Physiology with a minor in Data Science after receiving his MS and BS in Exercise Science from Indiana State University where he was also an athlete and coach.His interests vary widely but involve improving understanding of sport performance through athlete assessment, training, nutrition, environmental physiology, prediction modeling, and data mining sports data sets.He also learned web development to make his dissertation project, an interactive altitude adjustment calculator for track and field events, available to the public at trackcalculator.com.

Figure 1 .
Figure 1.Schematic of the randomized, double-blind, and parallel group study design.

Figure 2 .
Figure 2. ConSort flow chart.this figure shows the flow of subjects through the trial according to the criteria recommended in the ConSort guidelines.

Figure 5 .
Figure 5. maximal isometric voluntary contraction in percent change response to the 10 × 10 eccentric back squat protocol.*prWp and placebo groups significantly different pre-eimD to 24h, # prWp significantly different at 72h compared to pre-eimD.

Figure 7 .
Figure 7. Countermovement jump percent change in response to the 10 × 10 eccentric back squat protocol.**placebo group significantly different from all other groups at 72h.

Figure 6 .
Figure 6.peripheral fatigue in percent change response to the 10 × 10 eccentric back squat protocol.*prWp and placebo groups significantly different pre-eimD to 24h.

Figure 8 .
Figure 8. Barbell back squat velocity in percent change response to the 10 × 10 eccentric back squat protocol.*CWp and placebo significantly different at 24h visit.**prWp and CWp significantly different between pre-eimD and 24h.# placebo group significantly different between 24 and 72h.
Dr. Matthew J. Barenie, PhD, RD, is a Post-Doctoral Fellow within the Center for the Study of Obesity at the University of Arkansas for Medical Sciences.He received his BS in Dietetics from Indiana University, MS in Nutrition and Physical Performance at Saint Louis University, and his PhD in Exercise Physiology from Indiana University.Being a Registered Dietitian, his main research interests include optimizing physical performance and health through nutrition, training, and dietary supplementation.
Dr. Stephen J. Carter, MS, PhD, is an Assistant Professor in the Department of Kinesiology at Indiana University Bloomington.As a cardiovascular physiologist, Dr. Carter's research interest resides at the intersection of advancing age, obesity, and the menopausal transition.His work often employs multiple non-pharmacologic approaches including exercise training to enhance biomarkers of cardiometabolic health and physical function.An ongoing study is testing the utility of pre-exercise dietary nitrate to circumvent the constraints of exercise adaptation in late postmenopausal women.Ms.Hope E. Grange, BS, is a Health and Wellness Coach with WebMD Health Services specializing in the coaching of chronic health conditions.She received her BS in Fitness and Wellness from the School of Public Health at Indiana University.She is a certified ACE Health Coach and versatile group fitness instructor.Dr.Hunter L. Paris, PhD, is an exercise physiologist and an Associate Professor of Sports Medicine at Pepperdine University.His research centers around the interplay between exercise, energy balance, and chronic disease, aiming to better understand and improve long-term maintenance of lost body weight.His recent work has investigated how the high altitude environment influences metabolic rate and energy metabolism in health and disease.Ms.Danielle Krinsky, BS, is a Master of Public Health candidate at SUNY Downstate Health Sciences University.She received her BS degree in Exercise Science at Indiana University Bloomington.She has served as an undergraduate research assistant in the Exercise Physiology department during her time at Indiana University.Ms. Abigail S. Sogard, BSK, is a Master's and PhD student in Human Performance at Indiana University.She received her BSK degree in Exercise Science and Nutrition from Indiana University.

Table 2 .
Subjects' dietary intake before and during the experimental period.