Soy protein supplementation is not androgenic or estrogenic in college-aged men when combined with resistance exercise training

It is currently unclear as to whether sex hormones are significantly affected by soy or whey protein consumption. Additionally, estrogenic signaling may be potentiated via soy protein supplementation due to the presence of phytoestrogenic isoflavones. Limited evidence suggests that whey protein supplementation may increase androgenic signalling. Therefore, the purpose of this study was to examine the effects of soy protein concentrate (SPC), whey protein concentrate (WPC), or placebo (PLA) supplementation on serum sex hormones, androgen signaling markers in muscle tissue, and estrogen signaling markers in subcutaneous (SQ) adipose tissue of previously untrained, college-aged men (n = 47, 20 ± 1 yrs) that resistance trained for 12 weeks. Fasting serum total testosterone increased pre- to post-training, but more so in subjects consuming WPC (p < 0.05), whereas serum 17β-estradiol remained unaltered. SQ estrogen receptor alpha (ERα) protein expression and hormone-sensitive lipase mRNA increased with training regardless of supplementation. Muscle androgen receptor (AR) mRNA increased while ornithine decarboxylase mRNA (a gene target indicative of androgen signaling) decreased with training regardless of supplementation (p < 0.05). No significant interactions of supplement and time were observed for adipose tissue ERα/β protein levels, muscle tissue AR protein levels, or mRNAs in either tissue indicative of altered estrogenic or androgenic activity. Interestingly, WPC had the largest effect on increasing type II muscle fiber cross sectional area values (Cohen’s d = 1.30), whereas SPC had the largest effect on increasing this metric in type I fibers (Cohen’s d = 0.84). These data suggest that, while isoflavones were detected in SPC, chronic WPC or SPC supplementation did not appreciably affect biomarkers related to muscle androgenic signaling or SQ estrogenic signaling. The noted fiber type-specific responses to WPC and SPC supplementation warrant future research.


Rationale & background information
There is widespread evidence suggesting protein supplementation enhances resistance training adaptations. For instance, Cribb et al. 1 reported that resistance trained participants who consumed 45 g/d of whey protein isolate following 10 weeks of resistance training achieved a 5 kg increase in lean body mass (LBM) which was 4.2 kg greater than a casein-supplemented group. Burke et al. 2,3 reported that whey protein supplementation promoted 2.3-2.5 kg increases in LBM with 6 weeks of resistance training which was ~1.4 kg greater than the effects observed in these studies' maltodextrin placebo (PLA) groups. Notably, a soy protein group was also examined in one of the two aforementioned studies 3 , and this group experienced a 1.7 kg increase in LBM which was not statistically greater than the PLA group. Hulmi et al. 4 reported that 10.5 weeks of resistance training and whey protein supplementation elicited a 2.5 kg increase in LBM which was 0.5 kg greater than the non-energetic PLA group. Furthermore, Volek et al. 5 reported that 9 months of resistance training combined with whey or soy protein supplementation resulted in 3.6 and 2.6 kg increases in LBM, respectively. While the aforementioned studies did not determine the mechanisms responsible for the reported phenotypic changes in skeletal muscle mass, others have postulated that protein supplementation reduces fast to slow isoform shifts 6 and promotes myogenic responses to resistance training 7 . A recent review by Morton et al. 8 provides additional studies reporting that protein supplementation in conjunction with resistance training enhances indices of skeletal muscle anabolism, although other studies have reported that protein supplementation has no added benefit when performed in conjunction with 8-12 weeks of resistance training 9,10 .
Contrary to much of the positive data supporting whey protein supplementation, the data appears to be less favorable regarding the effects of amino acid-only supplementation in enhancing resistance training induced increases in LBM. For instance, Bird et al. 11 reported that essential amino acid (EAA) supplementation (6 g/d) led to ~3 kg increase in LBM following 12 weeks of resistance training twice a week in younger untrained males, albeit these increases were not significantly different from a PLA group. Vieillevoye et al. 12 reported similar findings in younger untrained males whereby 15 g/d of EAA supplementation during 12 weeks of resistance training did not increase LBM compared to a sucrose PLA group. Additionally, Aguilar et al. 13 recently reported that younger male subjects who supplemented with L-leucine (LEU; 3 g/d) during 8 weeks of resistance training experienced no additional increase in quadriceps muscle size increases when compared to subjects consuming a cornstarch PLA.
In spite of the aforementioned literature suggesting whey or soy protein supplementation may be more effective than EAA or LEU in promoting additional increases in LBM with resistance training, a prevailing hypothesis is that the LEU content of a given dietary protein determines the efficacy of how that protein potentiates muscle growth. However, this hypothesis is based on acute human, animal, or cell culture-based studies reporting that LEU or whey protein (which contains 8-11% LEU) optimally stimulates muscle protein synthesis [14][15][16][17] . To this end, there is no evidence which has directly compared the anabolic effects of LEU supplementation versus supplementation with other dietary protein sources that contain high levels of LEU (e.g., whey or soy).

Study goals and objectives
The purpose of this study was to examine if supplementation with LEU, whey protein concentrate (WPC), whey protein hydrolysate (WPH) or soy protein concentrate (SPC) enhances markers of skeletal muscle hypertrophy with resistance training in previously untrained, collegeaged males. A secondary aim was to also assess how these different supplements affected subcutaneous (SQ) fat cell size from biopsy specimens given that recent data from our group has demonstrated that WPH can elicit lipolytic effects 10,18,19 . Notably, the servings from all supplement groups (except the maltodextrin placebo described below) were standardized for LEU content (~3 grams per serving) and ingested twice daily.

Study design
The study design implemented was double-blinded and placebo-controlled ( Figure 1). Participants were encouraged to refrain from rigorous physical activity for 4-5 days prior to baseline testing (T1). For T1, participants were instructed to report to the laboratory in a wellhydrated, 4-hour fasted state whereby they were subjected to the following assessments: a) urine specific gravity, b) height and body mass, c) body composition using dual-energy X-ray absorptiometry (DXA) (General Electric Lunar Prodigy enCORE, software version 10.50.086; Madison, WI, USA), d) vastus lateralis thickness using ultrasonography (General Electric LOGIQ S7 Expert; Chicago, IL, USA), e) venipuncture, f) percutaneous skeletal muscle biopsy collection from the vastus lateralis, and g) a percutaneous SQ fat biopsy from the gluteal region. Two to three days following T1, subjects reported back to the laboratory in a 4-hour fasted state for a second visit (T2) whereby maximal force production capacity was assessed using an isometric mid-thigh pull (IMTP) test, lower body strength was assessed using a three repetition maximum (3-RM) squat, and upper body strength was assessed using a 3-RM bench press. Additionally, during T2, subjects were familiarized with all lifts that were to be performed during the training intervention. Following T2, subjects engaged in 12 weeks of resistance training and supplementation. The last training bout (T38) consisted of IMTP as well as squat and bench press 3-RM re-assessments in a 4-hour fasted state. Seventy two hours following T38, subjects reported back to the laboratory in a 4-hour fasted state for post-testing (T39) which consisted of all body composition, and blood and biopsy collection procedures noted for T1. All of the aforementioned testing procedures as well as training and supplementation procedures are described in greater detail below.  Abbreviations: DXA, dual x-ray absorptiometry; 3-RM, three-repetition maximum test; IMTP, isometric mid-thigh pull.

Body Composition Testing
During T1 and T39 participants were instructed to submit a urine sample (~5 mL) to assess normal hydration specific gravity levels (1.005-1.020 ppm) using a handheld refractometer (ATAGO; Bellevue, WA, USA). Participants with a urine specific gravity >1.020 were asked to consume tap water every 15 min for 30 min and then were re-tested. Following hydration testing, height and body mass were assessed using a digital column scale (Seca 769; Hanover, MD, USA) with weights and heights collected to the nearest 0.1 kg and 0.5 cm, respectively. Next, participants were subjected to a full body DXA scan while wearing general sports attire (i.e., athletic shorts or compression shorts and an athletic shirt) to assess various body composition characteristics. Dual arm and dual leg lean muscle mass, as assessed by the accompanying software, were used to estimate total body skeletal muscle mass (TBMM) by employing the equation from Kim et al. 20 , as reported by our group previously 10 . Notably, body segmentation for each scan was standardized prior to analyses by the same technician. Total body fat mass was also assessed by the accompanying software. According to previous data published by our laboratory, the same-day reliability of the DXA during a test-calibrate-retest on 10 participants produced intra-class correlation coefficients of 0.998 for total body fat mass (mean difference between tests = 0.40±0.05 kg), 0.998 for total body lean mass (mean difference between tests = 0.29±0.13 kg), and 0.998 for dual-leg lean mass (mean difference between tests = 0.17±0.09 kg) 21 .
Following DXA scans, participants were subjected to an ultrasound assessment to determine vastus lateralis muscle thickness. Measurements were taken from the midway point between the iliac crest and patella of the right femur whereby subjects were in a standing position and all weight was placed on the left leg. All DXA scans and ultrasound assessments were completed by the same investigator as suggested by previous research interventions 10,22 in order to minimize variability in testing procedures.

Venipuncture, and percutaneous skeletal muscle and fat biopsies
T1 and T39 venous blood samples were aseptically collected from the antecubital vein and collected into a 5 mL serum separator tube (BD Vacutainer; Franklin Lakes, NJ, USA). Notably, this blood was saved for further experimentation and variables assessed from these blood draws are not presented herein. Immediately following blood collection, participants were instructed to lay in a supine position on a treatment table whereby a percutaneous skeletal muscle biopsy was aseptically obtained from the left vastus lateralis muscle using a 5 gauge Bergstöm needle with suction as previously described by our laboratory [23][24][25][26] . Approximately, 20-40 mg of skeletal muscle tissue for each time point was placed in a cryomold with OCT media (Electron Microscopy Sciences; Hatfield, PA, USA). Cryomolds were then slow-frozen in liquid-nitrogencooled isopentane and stored at -80°C for immunohistochemistry analyses that are described below. Sections of SQ fat (1-2 cm) extracted from the gluteal aspect of the left hip were placed in 10% formalin and preserved for hematoxylin and eosin (H&E) staining and histological analyses which are described in detail below. Following T1 testing procedures, subjects were counterbalanced into one of five groups based upon DXA LBM values in order to ensure that baseline values did not differ between supplement groups. More details regarding supplementation are described below, and supplementation began immediately following the first training bout (T3).

Isometric mid-thigh pull, strength testing, and weightlifting familiarization
During T2 and T38, participants were instructed to report back to the laboratory under well hydrated, 4-hour fasted conditions for strength testing and weight training familiarization (T2). First, each participant completed an IMTP test which has been validated to approximate whole-body maximal voluntary strength [27][28][29] . Briefly, knee and hip angles (125±5° and 175±5°, respectively) were measured using a standard goniometer (Fabrication Enterprises; White Plains, NY, USA). A standard, 20 kg barbell (York Barbell; York, PA, USA) and STS Power Rack (York Barbell) were used to conduct the IMTP. Dual OR6-7 force plates (AMTI; Watertown, MA, USA) with dual Gen 5 amplifiers (AMTI) sampling at 500 Hz were used to measure vertical force production in Newtons (N). Similar to other investigations [29][30][31][32] , each participant was allowed at least two attempts, and up to four attempts if differences in vertical peak force between trials was >250 N. Manufacturer software was used to calculate vertical peak force during the testing sessions, and a custom-written MATLAB script (Natick, MA, USA) was employed to identify the greatest vertical force produced in N for each trial, post-hoc. Two trials within 250 N were used to calculate an average vertical peak force across trials and functioned as a metric for maximal voluntary force production (i.e., strength) in this investigation.
Approximately 5 min following T2 and T38 IMTP testing, participants performed 3-RM back squat and bench press assessments using a 20 kg barbell (York Barbell), STS Power Rack (York Barbell) and free weights. The demonstration of proper technique as well as the implementation of progressively-loaded 3-RM tests were overseen by C.B.M. and C.T.H. who possess the Certified Strength and Conditioning Specialist (CSCS) credential from the National Strength and Conditioning Association (NSCA). Bench press and back squat repetitions were considered to be successful when performed through the full range of motion (i.e., chest touch to full arm extension for bench press, and eccentric lowering past 90° knee flexion for back squat). A repetition was not counted if subjects exhibited poor and/or unsafe technique or needed assistance with a repetition during maximal testing.
Approximately 5 min following T2 3-RM testing, participants were instructed to perform the other two major lifts that were implemented for training (i.e., deadlift and bent-over-row) in the presence of CSCS-certified personnel. The goal of this session was to familiarize each participant with appropriate lifting technique in order to minimize the risk of injury throughout the course of the study.

Training protocol
For visits 3-37 (T3-T37), a daily undulating periodization (DUP) training model was employed over the 12-week training period given that this model has been shown to be more beneficial in eliciting greater increases in strength 33,34 and hypertrophy 35,36 than traditional linear periodization training models. Specifically, participants were instructed to perform freeweighted barbell squats, bench press, deadlifts, and bent-over-rows for 4 sets of 10 repetitions (Monday or Tuesday), 6 sets of 4 repetitions (Wednesday or Thursday), and 5 sets of 6 repetitions (Friday or Sunday). Immediately following each completed set, a rating of perceived exertion (RPE) score was acquired from each participant (scale: 1-10) in order to monitor and progress each participant accordingly while minimizing the potential risk of injury [37][38][39][40] . The RPE scale was described to participants as the remaining number of repetitions that the participant would be able to complete while employing good technique (i.e., 1 = 9 remaining repetitions in reserve, 10 = 0 remaining repetitions in reserve). More information on relative training intensities and progression can be found in Table 1. Participants were instructed to attend all 36 resistance training sessions throughout the duration of the study, but those that missed more than 4 sessions were not included in the analysis due to lack of training compliance. All participants were supervised by laboratory personnel for each training session to ensure that proper lifting technique was executed, and training volumes for each session were recorded.

Supplementation
As stated above, participants were assigned to ingest either a PLA, LEU, WPC, WPH, or SPC supplement throughout the training intervention. More information regarding the macronutrient profile for a serving size of each supplement can be found in Table 2. On training days (T3-T37), participants consumed an individually-packaged serving in ~500 ml of tap water immediately following each training session under direct observation of the study personnel. Additionally, participants were instructed to consume an individual serving within 30 min prior to bedtime on training days given that this strategy has been shown to be effective for stimulating overnight muscle protein synthesis 41 . On non-training days, participants were instructed to consume an individual serving between a meal of their choosing and 30 min prior to bedtime. Supplements were separated into individual ready-made supplement-coded packets for daily consumption, and participants were given a 3-week supply. Study personnel collected and counted empty packets from each participant every 3 weeks before the next 3-week supply was distributed. Participants that did not consume >80% were not included in the analysis due to lack of compliance.
Each supplement, except PLA, was formulated to provide ~3 g of leucine, per serving. Furthermore, each supplement was formulated to yield similar amounts of total energy (kcal) and fat (g), and was double-blinded to laboratory personnel and participants for group, appearance, taste, texture, and packaging. The WPC supplement was formulated using an agglomerated, 80% WPC (Hilmar™ 8010, Hilmar Ingredients; Hilmar, CA, USA). The WPH supplement was formulated using an agglomerated, partially hydrolyzed [12.5% degree of hydrolysate (12.5% DH), yielding approximately 67% of peptides as <5 kilodaltons (kDa) in molecular weight] 80% whey protein concentrate (Hilmar™ 8360, Hilmar Ingredients); SPC used an agglomerated, 80% soy protein concentrate (ALPHA® 5812, Solae, LLC; St. Louis, MO, USA); LEU used an agglomerated, L-Leucine (L-Leucine USP, Glambia Nutritionals; Carlsbad, CA, USA) and non-GMO, corn-derived maltodextrin (MALTRIN®-M100; Grain Processing Corporation; Muscantine, IA, USA); and, the PLA group was formulated using maltodextrin (MALTRIN®-M100; Grain Processing Corporation). All five supplements were manufactured at JW Nutritional, LLC (Allen, TX, USA), a United States Food and Drug Administration cGMPcompliant facility independently audited and pre-qualified by Obvium*Q, LLC (Phoenix, AZ, USA), a GMP regulatory compliance firm. Personnel at JW Nutritional, LLC and C.M.L. (Lockwood, LLC; Draper, UT, USA) formulated and maintained blinding of groups, and each supplement was assigned a randomly generated item number. Manufacturing batch records for production of each of the five supplements were reviewed by a trained, independent expert in dietary supplement quality control and assurance (C.M.L.) before approval for use within the present study. All supplements were independently validated for nutritional facts and total amino acids using validated, approved methods at Covance Laboratories, Inc. (Madison, WI, USA), a pre-qualified third-party analytical laboratory, and results reviewed by C.M.L. prior to the supplements being approved for use within the present study. Once analysis was complete, a Lockwood, LLC representative not involved in the study released the code for all treatments. Abbreviations: PLA, maltodextrin placebo; LEU, L-leucine; WPC, whey protein concentrate; WPH, whey protein hydrolysate; SPC, soy protein concentrate; g, grams; mg, milligrams; IU, international units; kD, kilodaltons.

Nutritional intake monitoring
Participants were instructed to maintain their normal dietary habits along with returning a 4-day food log (2 week days and both weekend days) at baseline (T1), week 6 (T20) and week 12 (T39). On each occasion, participants were given detailed written and verbal instructions on completing the food logs. Dietary intake data were analyzed using open-sourced software (http://www.myfitnesspal.com), which has been employed to analyze food intake data in other studies [42][43][44][45][46][47][48] .

Safety considerations
In the event that a subject suffered an injury from training, this was immediately reported to Dr. Michael Goodlett (MD) who is the medical oversight of the Auburn University IRB. Additionally, any biopsy-related issues (e.g., hematomas) were reported to Dr. Goodlett. One participant did sustain an injury during this study, and he was referred to Dr. Goodlett. Additionally, this adverse event was filed with the Auburn IRB. This individual has since recovered from this injury.

Follow-up
Following the conclusion of training, the subjects were no longer required to report to the laboratory unless they had issues from the biopsy sites (e.g., hematoma formation or swelling). Notably, there were no adverse events related to biopsies, and no subjects reported adverse events related to being a participant in the study.

Data Management and Statistical Analysis
For the current paper submitted to Scientific Reports analyzing the SPC, WPC and PLA groups statistical tests were performed in RStudio (Version 1.0.143) and SPSS (Version 23). The average sample size for each dependent variable was 33 subjects, while specific sample sizes for each analysis are reported in figures or text. Group (3 levels [PLA, SPC, WPC]) and time (2 levels [PRE, POST]) served as independent variables in this analysis. Measurement of each dependent variable occurred at each time point. A-priori power analyses in RStudio using general linear model parameters in the "pwr" package (Version 1.2-1) revealed 79 % power (power = 1β) for the discovery of a medium size effect, and 98 % power for the discovery of a large effect when analyses included 33 subjects with 2 observations (i.e., 66 total observations). Supplementary tables provide descriptive statistics for each dependent variable with no outlier removal, mean differences, Cohen's d effect sizes, and 95 % confidence intervals. Data are not restated in results when included in figures. Data are reported as mean ± standard error of the mean, and are based on any data removal described below. The alpha level of significance was set a-priori to: p ≤ 0.050. Statistical assumptions tests were completed prior to statistical analysis consisting of: a) Shapiro-Wilks tests of residual distributions for normality, and b) Levene's test of homogeneity of variance, given that a repeated measures analysis of variance (ANOVA) was performed for the provision of p-values. Violation of these assumptions are reported and appropriate data transformations (i.e., square root or log10 transformations) were completed when residuals were not normally distributed prior to ANOVA for the avoidance of type 1 or type 2 errors. Data transformation and data removal were avoided with intention to report raw data and associated inferential statistics. For this reason, if the majority of levels of group (2 of 3 groups) at each level of time were normally distributed, ANOVA proceeded. However, if datum met both of the following two criteria, datum were removed to best reflect group responses and more confidently infer effects: 1) Shapiro-Wilks tests revealed non-normal distributions for the data array and, 2) the datum was ≥ 3 standard deviations from the group mean. Although rare, cases of data removal were reported. For significant group × time interactions, two-tailed independent samples t-tests were performed as a post-hoc test. For significant time F-statistics identified from ANOVA, a 2-tailed dependent samples t-test was performed comparing PRE to POST data arrays.

Quality assurance
All data obtained from training was kept in de-identified participant folders with study codes (e.g., "Subject 1"). Data entry was conducted by laboratory personnel, and data entry was double-checked by other laboratory personnel along with the principal investigator. Data entry from biochemical assays were entered by a graduate student, and these data were double-checked by the principal investigator. Form must be populated using Adobe Acrobat / Pro 9 or greater standalone program (do not fill out in browser). Hand written forms will not be accepted.

Protocol Number:
15-320 MR1508  None, we are beginning this study this upcoming summer. This document is titled 'Proposed amendments for Protein study (15-320 MR1508)'

Identify any changes in the anticipated risks and / or benefits to the participants.
Additional anticipated risks: a) more 'soreness' from a full-body workout versus a leg workout, b) more soreness from the two additional fat biopsies Additional anticipated benefits: compensation of $500 versus $300 11. Identify any changes in the safeguards or precautions that will be used to address anticipated risks.
Participants will be progressively trained (i.e., will not start with heavy weight-lifting, but will use lighter lifting loads and build up slowly to more challenging loads). Moreover, participants can discontinue the study if the workouts make them too sore. The same precautions will be taken with fat biopsies as with the muscle biopsies. Again, the participants can discontinue the study if the biopsies make them too sore. However, we have not had this issue in other IRB-approved studies with muscle biopsies and do not anticipate this being an issue with the fat biopsies.
12. Attach a copy of all "stamped" IRB-approved documents you are currently using. (information letters, consents, flyers, etc.)

of 2
Question #9: AMENDMENT REQUEST #1 (reducing the number of participants) The original purpose of the study was to determine muscle-building effects of supplementing with either 2 doses per day (dose amounts are in parentheses) of: • whey protein concentrate (40 g/day twice daily) (n=15 participants per group were to be recruited) • whey protein hydrolysate (40 g/day twice daily), (n=15 participants per group were to be recruited) • egg protein concentrate (40 g/day twice daily), (n=15 participants per group were to be recruited) • egg protein hydrolysate (40 g/day twice daily), (n=15 participants per group were to be recruited) • micellar casein (40 g/day twice daily), (n=15 participants per group were to be recruited) • soy protein concentrate (40 g/day twice daily), (n=15 participants per group were to be recruited) • branched chain amino acids (6 g/day twice daily), (n=15 participants per group were to be recruited) • and a maltodextrin placebo (6 g/day twice daily), (n=15 participants per group were to be recruited) n=120 participants were to be recruited We now only wish to test the following groups (not crossed out): • whey protein concentrate (40 g/day twice daily), • whey protein hydrolysate (40 g/day twice daily), • egg protein concentrate (40 g/day twice daily), • egg protein hydrolysate (40 g/day twice daily), • micellar casein (40 g/day twice daily), • soy protein concentrate (40 g/day twice daily), • branched chain amino acids (6 g/day twice daily), • and a maltodextrin placebo (6 g/day twice daily) n=75 participants will now be recruited, although with potential attrition we expect to recruit 120 participants.

Rationale:
We are unable to procure egg and casein protein from industry sponsors. Notwithstanding, this study will still compare different whey protein supplements versus soy and amino acids supplements, and has extremely valuable implications in terms of which supplement is optimal for improving body composition.
AMENDMENT REQUEST #2 (alteration of the workout for participants) Below is the original study design that is currently approved: Each gray arrow is a workout to be completed in the School of Kinesiology. Each workout was originally 4-6 sets of leg extensions for 4-10 repetitions. Below shows the leg extensor exercise for frame of reference: We now request that each workout be a 'full-body' (or 'whole-body') workout which will include: -3 sets of squats for 3-10 repetitions -3 sets of bench press for 3-10 repetitions -3 sets of bent-over rows for 3-10 repetitions -3 sets of deadlifts for 3-10 repetitions -3 sets of crunches for 3-10 repetitions Below is a diagram of each exercise:

Squat
Bench press Rows Crunches

Deadlift
Rationale: The purpose of this request is to utilize a whole-body workout that has more beneficial whole-body effects rather than just focusing on the leg muscles with the originallyproposed leg extensor training. This will better decipher if protein or amino acid supplementation affects whole-body composition versus just leg musculature.

AMENDMENT REQUEST #3 (obtaining 2 fat biopsies)
Below is the original study design that is currently approved: At T1, T3 and T39, there are muscle biopsies that are currently approved. We now wish to also obtain a fat biopsy from the leg as well at T1 and T39.
This process is very similar to the muscle biopsy in that it involves administering lidocaine to the collection site, making a pilot incision with a sterile blade, and placing the biopsy needle in the collection site. However, unlike the muscle biopsy, we will not penetrate the collection site as deep and only collect superficial adipose tissue. Below is a schematic that shows the differences between the two techniques: Importantly, fat biopsies are equally as safe as muscle biopsies. Moreover, Dr. Roberts has performed over 200 muscle biopsies to date here on Auburn's campus with zero post-biopsy adverse events being reported. Dr. Roberts had a meeting with Dr. Mike Goodlett about the fat biopsy procedure to ensure that Dr. Goodlett was fully aware of this proposed procedure.
The particular collection site will be the upper buttock region. Specifically, Dr. Roberts will sterilize the area as discussed with the muscle biopsy procedure and administer 1 cc of lidocaine. Dr. Roberts will then pinch a large area of fat in this area, make a pilot incision with a sterile blade, and penetrate the biopsy needle superficially in order to obtain a pencil erasuresize amount of fat tissue. Thereafter, a butterfly Band-Aid and large elastic bandage with gauze will be placed over the biopsy site. As with the 24-h checkup on the muscle biopsy, participants will have the fat biopsy collection site inspected as well.
Rationale: Whey protein supplementation has been shown to reduce body fat. Samples collected from the fat biopsy technique will be histologically examined in order to observe if fat cell size is reduced with whey protein supplementation versus the other supplements. Moreover, the fat samples will also be analyzed for the genetic expression of 'fat-building' genes. Our working hypothesis is that whey protein will be superior to soy protein as well as amino acid supplementation on reducing fat cell size and the expression of 'fat-building' genes.
AMENDMENT REQUEST #4 (subject compensation) The original protocol approved subjects to be compensated $300. We now wish to pay participants $500 upon completion of the study. A new partial compensation will be as follows: • $50 for completion of visits 1-2 (1 fat biopsy and 1 muscle biopsy given) • An additional $100 for completion of visits 1-3 (1 fat biopsy and 2 muscle biopsies given) • An additional $200 for completion of up to all visits except visit #39 (meaning that all workouts were completed but the last testing session was missed and, thus, the last muscle and fat biopsy was not taken) • An additional $150 for completion of visit #39 Given that we are wishing to implement a more involved workout (full-body versus leg extensions only) and are wishing to collect 3 muscle biopsies (originally-approved) as well as two fat biopsies (AMENDMENT REQUEST #3 above), we feel that this is a fair compensation for participants.

Opportunity to Participate in a Paid Research Study
Titled:

The Effects of Different Protein and Amino Acid Supplements on Muscle Hypertrophy after 12 Weeks of Resistance Exercise in Untrained Men
"Researchers in The School of Kinesiology at Auburn University are conducting a study which will evaluate which amino acid or protein source supplement (specifically branched chain amino acids, whey, or soy) enhances markers of skeletal muscle growth and body fat reduction when ingested in conjunction with chronic resistance training.
You may qualify if you: 1) are between the ages of 19-35 and have a body mass index (body mass in kilograms/ height in meters^2) between 23-30 2) are apparently healthy and do not have any known illnesses that are contraindicated for exercise (i.e., hypertension, arrhythmias, or cardiopulmonary diseases), or are not taking medications for these diseases 3) have not engaged in weight training over the past 12 months; this means less than one training session per every two weeks 4) have not engaged in lower-body endurance training over the past 12 months; this means that the participant has not run more than 10 miles per week or road-biked/swam more than 2 days per week 5) have not consumed nutritional supplements two months prior to the study including hormone boosters, protein supplements, amino acid supplements and/or creatine monohydrate 6) are not regular tobacco users, or haven't been over the past 12 months 7) do not have any blood clotting issues and/or are on blood-thinning agents or medications If you decide to participate in this research study, you will be asked to: 1) Complete a consent and pre-test at the Auburn School of Kinesiology, located at 301 Wire Road, Auburn AL (room 136), lasting 80 min, where you will perform a whole-body x-ray scan, ultrasound scan of the leg, blood draw, and skeletal muscle biopsy from the mid-thigh and fat biopsy from the upper buttock region. 2) Complete visit #2, lasting 60 min, which involves doing a leg strength test (BIODEX), and practice leg exercise session. 3) Complete visit #3, lasting 4 hours, where you will perform a whole-body resistance training workout and in the Kinesiology building and donate a 2 nd muscle biopsy from the mid-thigh as well as two additional blood draws. 4) Complete visits 4-38 over a 12-week period, lasting 45 min each, where you will perform whole-body resistance training workouts and in the Kinesiology building. 5) Complete a visit #39, lasting 80 min, where you will perform a whole-body x-ray scan, ultrasound scan of the leg, blood draw, a 3 rd skeletal muscle biopsy from the mid-thigh, a 2 nd fat biopsy from the upper buttock region and BIODEX.
The total approximate time commitment in the Kinesiology building will be approximately 33-36 hours.
You will be monetarily compensated in the form of a $500 check for your time commitment upon completion of the study (partial compensation will be provided for non-completion).
If you are interested in participating in this study, please e-mail your name and contact information to Brooks

Advertisement for a Paid Research Study Titled: The Effects of Different Protein and Amino Acid Supplements on Muscle Hypertrophy after 12 Weeks of Resistance Exercise in Untrained Men
Researchers in The School of Kinesiology at Auburn University are conducting a study which will evaluate which amino acid or protein source supplement (specifically branched chain amino acids, whey, or soy) enhances markers of skeletal muscle growth and body fat reduction when ingested in conjunction with chronic resistance training.
You may qualify if you: 1) are between the ages of 19-35 and have a body mass index (body mass in kilograms/ height in meters^2) between 23-30 2) are apparently healthy and do not have any known illnesses that are contraindicated for exercise (i.e., hypertension, arrhythmias, or cardiopulmonary diseases), or are not taking medications for these diseases 3) have not engaged in weight training over the past 12 months; this means less than one training session per every two weeks 4) have not engaged in lower-body endurance training over the past 12 months; this means that the participant has not run more than 10 miles per week or road-biked/swam more than 2 days per week 5) have not consumed nutritional supplements two months prior to the study including hormone boosters, protein supplements, amino acid supplements and/or creatine monohydrate 6) are not regular tobacco users, or haven't been over the past 12 months 7) do not have any blood clotting issues and/or are on blood-thinning agents or medications If you decide to participate in this research study, you will be asked to: 1) Complete a consent and pre-test at the Auburn School of Kinesiology, located at 301 Wire Road, Auburn AL (room 136), lasting 80 min, where you will perform a whole-body x-ray scan, ultrasound scan of the leg, blood draw, and skeletal muscle biopsy from the mid-thigh and fat biopsy from the upper buttock region. 2) Complete visit #2, lasting 60 min, which involves doing a leg strength test (BIODEX), and practice leg exercise session. 3) Complete visit #3, lasting 4 hours, where you will perform a whole-body resistance training workout and in the Kinesiology building and donate a 2 nd muscle biopsy from the mid-thigh as well as two additional blood draws. 4) Complete visits 4-38 over a 12-week period, lasting 45 min each, where you will perform whole-body resistance training workouts and in the Kinesiology building. 5) Complete a visit #39, lasting 80 min, where you will perform a whole-body x-ray scan, ultrasound scan of the leg, blood draw, a 3 rd skeletal muscle biopsy from the mid-thigh, a 2 nd fat biopsy from the upper buttock region and BIODEX.
The total approximate time commitment in the Kinesiology building will be approximately 33-36 hours.
You will be monetarily compensated in the form of a $500 check for your time commitment upon completion of the study (partial compensation will be provided for non-completion).
If you are interested in participating in this study, please e-mail your name and contact information to Brooks Mobley at moblecb@auburn.edu or Mike Roberts, PhD at mdr0024@auburn.edu. 2) are apparently healthy and do not have any known illnesses that are contraindicated for exercise (i.e., hypertension, arrhythmias, or cardiopulmonary diseases), or are not taking medications for these diseases 3) have not engaged in lower-body weight training over the past 12 months; this means less than one training session per every two weeks 4) have not engaged in lower-body endurance training over the past 12 months; this means that the participant has not run more than 10 miles per week or road-biked/swam more than 2 days per week 5) have not consumed nutritional supplements two months prior to the study including hormone boosters, protein supplements, amino acid supplements and/or creatine monohydrate 6) are not regular tobacco users, or haven't been over the past 12 months Below is a schematic of the study timeline, and following the schematic is a detailed paragraph of each testing session:

Visit #1 (T1, 80 min)
You will report to Kinesiology Building room 286 for approximately 1 hour for: An initial consent, medical questionnaire, and post-consent testing (10 min). During this session you will consent to or decline participation. If you consent, you will fill out a brief medical history questionnaire and the PI or a lab member will explain the sequence of testing events for the study. After filling out these forms, you will then perform a series of tests below: Height & weight (5 min). You will be asked to remove your shoes and your height and weight will be measured using a standard balance scale. Urine specific gravity testing (5 min). You will then be given a disposable Dixie cup and be instructed to proceed to the restroom and urinate in the cup. You will then leave the cup on the urinal and a lab member will go into the bathroom and retrieve it to analyze the specific gravity using a refractometer. The purpose of this test is to ensure adequate hydration for the body composition testing.

Dual x-ray absorptiometry (DEXA) body composition testing (10 min).
You will then be walked downstairs to KINE room 125 and will have your body composition tested using a 'DEXA scanner'. The scan requires you to remain still for 7-10 minutes. During the scan, a low-dose x-ray beam will pass through the entire body. According to the scientific literature, the total radiation exposure is less than that of an airline flight from California to New York and back.
Please note these DEXA scans are NOT being conducted for clinical purposes which means they are not designed to assess any medical condition you may have. They are being conducted for research purposes only and are not designed to reveal any existing disease or pathology. If however your scan reveals any unexpected findings given your medical history, it will be reviewed by a physician and he will communicate to you if there is a need to follow up with your preferred physician.
Ultrasound assessment for leg muscle thickness (5 min). Following the DEXA test, you will be escorted to KINE room 260 for this test. This test will require you to lay down face-up on an athletic training table whereby ultrasound pictures will be taken of the outside portion of your upper thigh (the vastus lateralis muscle). This is done by placing a hand-held ultrasound probe on top of the skin, using a small amount of hypoallergenic transmission gel.

Blood draw (5 min).
Following height and weight measurement, you will be asked to sit in a phlebotomy chair and have blood drawn from a vein in the front of your elbow. Approximately 1 teaspoon (6 milliliters) of blood will be collected using sterile supplies and techniques. The site will be cleaned and bandaged following the blood draw and you will be given instructions to minimize bruising or discomfort. In addition, in the extraordinarily rate event that you require medical care following the blood draw, you will be referred to go to the on-campus medical clinic or a clinic in the Auburn area. Skeletal muscle biopsy (20 min). Following the blood draw, a skeletal muscle biopsy procedure will be performed.
In the rare event that you are allergic to Lidocaine or betadine/iodine please immediately tell Dr. Roberts.
The person collecting the biopsy (Dr. Roberts) will explain the procedure once more.
Dr. Roberts will then prepare your left middle/outer thigh for the collection of a muscle specimen. Below outlines the events involved with this procedure: 1. The outer aspect of the left mid-thigh will be shaven with electronic clippers in the main laboratory area of room 241. 2. You will then enter an isolated athletic training room within laboratory 241 and an athletic training table will be wiped down with 10% bleach and absorbent paper. You will then lie face up on the training table. 3. The person collecting the biopsy (Dr. Roberts) will then garb in a clean lab gown and sterile gloves. 4. 4 'dots' will be drawn with a Sharpie marker on you left leg to denote where the injection and incision sites are to be made (this will be referred to as the 'collection field'). 5. The shaven portion of the leg will be cleansed with sterile, single-use alcohol pads. 6. 0.5 cc of 2% Lidocaine will then be injected subcutaneously within the collection field to de-sensitize pain receptors using a very small needle. During this time, you may feel a slight 'burning' sensation due to the Lidocaine entering the sub-dermal layer. The drug will be allowed 5 min to take effect. 7. 0.5 cc of 2% Lidocaine will then be injected deeper within the collection field to further de-sensitize pain receptors. The drug will be allowed another 5 min to take effect. a. During this time, the collection field and 2 inches beyond the collection field will then be swabbed with betadine solution. b. A sterile drape will then be placed atop the leg. 8. After the 5-min waiting period as mentioned in #7, a sterile/single-use no. 11 blade will be used to make a 1 cm incision (the width of your pinky nail) in the center of the collection field in order to facilitate the procedure. a. Any immediate bleeding will be swabbed with sterile gauze, although excessive bleeding in this area of the leg usually does not occur. b. After use, the blade will be placed in a sharps biohazard container and the soiled gauze pads will be placed in a biohazard container. 9. A sterilized biopsy needle (attached to a 60cc syringe with polyethylene tubing) will then be inserted into the pilot hole and will be pierced through the fascia. Once the needle breaches the fascia, Drs. Roberts will instruct the graduate student holding the syringe to apply suction. Dr. Roberts will then apply an upward and downward motion to the needle which excises ~200-300 mg of muscle tissue (which is roughly the size of a no. 2 pencil eraser). During this aspect of the biopsy, you should not feel sharp pain due to the administered lidocaine and will only feel pressure from the biopsy needle. This portion of the procedure typically lasts 5-7 seconds. 10. The needle will be removed from the leg, large sterile gauze pads will be placed atop the pilot incision, and pressure will be held by the investigator for up to 10 min. a. The assisting graduate student will take the needle and remove the muscle tissue for preservation. b. The student will then begin sterilizing the biopsy needle using bleach and an autoclave (high pressure, high temperature) oven. 11. Following pressure application on the collection field, the pilot incision will be pinched shut and a butterfly bandage will be applied to keep the pilot incision closed. 12. Triple antibiotic will be applied around the incision site, sterile gauze will be placed atop the butterfly bandage, and a large adhesive bandage will be placed atop the collection site.
Prior to leaving the laboratory, Dr. Roberts will hand you a biopsy care sheet to abide by in order to optimize wound closure from the procedure and ask that you return to the laboratory 24 hours after your testing session so that they can examine the collection sites. Fat biopsy (20 min). Following the skeletal muscle biopsy procedure, a fat biopsy will be performed.
In the rare event that you are allergic to Lidocaine or betadine/iodine please immediately tell Dr. Roberts.
The person collecting the biopsy (Dr. Roberts) will explain the procedure once more.
Dr. Roberts will then prepare the outer aspect of the upper right buttock (i.e., 'love handle'). Below outlines the events involved with this procedure: 1. You will remain on the isolated athletic training room within laboratory 241 after the muscle biopsy. You will then lie face down on the training table. 2. The person collecting the biopsy (Dr. Roberts) will then garb in a clean lab gown and sterile gloves. 3. 4 'dots' will be drawn with a Sharpie marker on your upper buttock to denote where the injection and incision sites are to be made (this will be referred to as the 'collection field'). 4. 0.5 cc of 2% Lidocaine will then be injected subcutaneously within the collection field to de-sensitize pain receptors using a very small needle. During this time, you may feel a slight 'burning' sensation due to the Lidocaine entering the sub-dermal layer. The drug will be allowed 5 min to take effect. 5. 0.5 cc of 2% Lidocaine will then be injected deeper within the collection field to further de-sensitize pain receptors. The drug will be allowed another 5 min to take effect. a. During this time, the collection field and 2 inches beyond the collection field will then be swabbed with betadine solution. b. A sterile drape will then be placed atop collection site. 6. After the 5-min waiting period, a sterile/single-use no. 11 blade will be used to make a 1 cm incision (the width of your pinky nail) in the center of the collection field in order to facilitate the procedure. a. Any immediate bleeding will be swabbed with sterile gauze, although excessive bleeding in this upper buttock region usually does not occur. b. After use, the blade will be placed in a sharps biohazard container and the soiled gauze pads will be placed in a biohazard container. 7. A sterilized biopsy needle (attached to a 60cc syringe with polyethylene tubing) will then be inserted into the pilot hole and will be pierced in an area of fat. Once the needle is settled, Drs. Roberts will instruct the graduate student holding the syringe to apply suction. Dr. Roberts will then apply an upward and downward motion to the needle which excises ~200-300 mg of fat tissue (which is roughly the size of a no. 2 pencil eraser). During this aspect of the biopsy, you should not feel sharp pain due to the administered lidocaine and will only feel pressure from the biopsy needle. This portion of the procedure typically lasts 5-7 seconds. 8. The needle will be removed from the collection site, large sterile gauze pads will be placed atop the pilot incision, and pressure will be held by the investigator for up to 10 min. a. The assisting graduate student will take the needle and remove the fat tissue for preservation. b. The student will then begin sterilizing the biopsy needle using bleach and an autoclave (high pressure, high temperature) oven. 9. Following pressure application on the collection field, the pilot incision will be pinched shut and a butterfly bandage will be applied to keep the pilot incision closed. 10. Triple antibiotic will be applied around the incision site, sterile gauze will be placed atop the butterfly bandage, and a large adhesive bandage will be placed atop the collection site.
Prior to leaving the laboratory, Dr. Roberts will hand you a biopsy care sheet to abide by in order to optimize wound closure from the procedure and ask that you return to the laboratory 24 hours after your testing session so that they can examine the collection sites.
Dr. Roberts or a staff member will also give you a 4-day food log to fill out prior to returning to the laboratory for T2 described below.
Visit #2 (T2, 60 min) 5-7 days following T1, you will report to Kinesiology room 286, and return your completed food log. You will then engage in the following tests that will last for approximately 1 hour: Isokinetic peak torque testing (15 min). You will then perform a knee extension peak isokinetic torque test of the right knee extensors. During this test you will have 1 warm-up set of 10 repetitions. Following the warm-up set, you will kick as forcefully as possible against a fixed speed (60 degrees per second) programmed by the BioDex System 4 Isokinetic dynamometer. This test involves extraordinarily low injury risk and is commonly used in rehabilitation settings in order to get a scientific measurement of leg strength.
Practice whole-body resistance exercise (40 min). You will then perform regular weight lifting exercises using a knee extension device that has resistance loaded with free weight plates. You will perform 1 sets of 3-10 repetitions of squats, bench press, bent-over rows, deadlifts and crunches in order to assess your strength. During this practice lifting session, we will determine your 'optimal' lifting load (or resistance) for which you can successfully lift for 3-5 times. This will serve to familiarize you with the exercise devices (which you'll use to train on for visits 3-38) as well as determine the training load to assign to you during these visits.

Assignment into a supplement or placebo group (5 min).
You will then be assigned to one of 5 groups described below and will be given a 12-week supply of your supplement. You will be blinded to the supplement composition. Supplements will include:

1.
Placebo (PLA). You will consume one serving of maltodextrin PLA capsules (6 g/serving) on whole body workout training days (described below which will occur during visits  immediately after each whole body workout and up to 1 hr prior to bedtime. On non-training days, you will be encouraged to consume one between meals and one serving up to 1 hr prior to bedtime.

2.
Branched-chain amino acids (BCAA). You will consume one serving of BCAA capsules (6 g/serving) on whole body workout training days (described below which will occur during visits  immediately after each whole body workout and up to 1 hr prior to bedtime. On non-training days, you will be Page 8 of 15 Participant's initials ______ encouraged to consume one between meals and one serving up to 1 hr prior to bedtime.

3.
Whey protein concentrate (WPC). You will consume one serving of WPC powder in 16 fl oz of water (40 g/serving) on whole body workout training days (described below which will occur during visits  immediately after each whole body workout and up to 1 hr prior to bedtime. On non-training days, you will be encouraged to consume one between meals and one serving up to 1 hr prior to bedtime.

4.
Whey protein hydrolysate (WPH). You will consume one serving of WPH powder in 16 fl oz of water (40 g/serving) on whole body workout training days (described below which will occur during visits  immediately after each whole body workout and up to 1 hr prior to bedtime. On non-training days, you will be encouraged to consume one between meals and one serving up to 1 hr prior to bedtime.

5.
Soy protein concentrate (SPC). You will consume one serving of SPC powder in 16 fl oz of water (40 g/serving) on whole body workout training days (described below which will occur during visits  immediately after each whole body workout and up to 1 hr prior to bedtime. On non-training days, you will be encouraged to consume one between meals and one serving up to 1 hr prior to bedtime.
You will then be scheduled for T3 which will occur 5-7 days following T2. We ask that you come at least 4 hours fasted to this session.

Visit #3 (T3, 4 hr)
You will report to Kinesiology Building room 286 for approximately 4 hours for:

Whole-body workout and supplement ingestion post-workout (45 min).
You will perform a whole-body workout which will consist of 3 sets of 3-10 repetitions of squats, bench press, bent-over rows, deadlifts and crunches (with 2 min of rest between sets). The lifting load (or resistance) will be 80-95% of your maximal effort as assessed previously during T2. The lifting load (or resistance) will be Page 9 of 15 Participant's initials ______ about 70-80% of your maximal effort which will have been determined from T2. Immediately after the exercise bout, you will ingest the supplement to which you were assigned to during T2.

Blood draws at 30 min and 60 min post-exercise (5 min).
Approximately 1 teaspoon (6 milliliters) of blood will be collected using sterile supplies and techniques described above at these two post-exercise time points. This will serve to examine if post-workout/post-supplement ingestion affects circulating serum amino acid levels.

2-h post-exercise biopsy skeletal muscle biopsy (20 min).
Following the blood draw, a skeletal muscle biopsy procedure will be performed on the left leg as described above. This will serve to examine if post-workout/post-supplement ingestion affects intramuscular anabolic signaling in an acute fashion after one bout and treatment.
More specifically, these acute blood and muscle responses will be examined to see if acute changes in these tissues are reflective of chronic changes with the 12-week protocol.

Visit #4-38 (T4 thru 38 which occur over a 12-week period, 45 min each)
You will report to Kinesiology Building room 136 for approximately 45 minutes for each visit for whole-body resistance training sessions. Typically, you'll come in on either Monday, Wednesday and Friday OR Tues, Thurs, and Saturday. You will perform 3 sets of 3-10 repetitions of squats, bench press, bent-over rows, deadlifts and crunches (with 2 min of rest between sets). The lifting load (or resistance) will be 80-95% of your maximal effort as assessed previously during T2. Following each training session (in the laboratory) we will have you consume one serving of your supplement to which you are assigned to. You will also be instructed to consume one serving of your supplement prior to bedtime. On non-training days, we ask that you consume your supplement once between meals and once prior to bedtime.
This phase of the study will occur over a 12-week period.

Visit #39 (T39, 80 min)
You will report to Kinesiology Building room 286 for approximately 1 hour for: Urine specific gravity testing (5 min). You will be given a disposable Dixie cup and be instructed to proceed to the restroom and urinate in the cup. You will then leave the cup on the urinal and a lab member will go into the bathroom and retrieve it to analyze the specific gravity using a refractometer. Again, the purpose of this test is to ensure adequate hydration for the body composition testing.

Dual x-ray absorptiometry (DEXA) body composition testing (10 min).
You will then be walked downstairs to KINE room 125 and will have your body composition tested using a 'DEXA scanner' as described above during T1.

Ultrasound assessment for leg muscle thickness (5 min).
Following the DEXA test, you will be escorted to KINE room 260 for this test as described above during T1.

Blood draw (5 min).
Following height and weight measurement, you will be asked to sit in a phlebotomy chair and have blood drawn from a vein in the front of your elbow. Approximately 1 tablespoon (6 milliliters) of blood will be collected as described above during T1.

Skeletal muscle biopsy (20 min).
Following the blood draw, a skeletal muscle biopsy procedure will be performed on the left leg as described above during T1.

Fat biopsy (20 min).
Following the blood draw, a fat biopsy procedure will be performed on the upper buttock as described above during T1.

Isokinetic peak torque testing (15 min).
You will then perform a knee extension peak isokinetic torque test of the right knee extensors. During this test you will have 1 warm-up set of 10 repetitions. Following the warm-up set, you will kick as forcefully as possible against a fixed speed (60 degrees per second) programmed by the BioDex System 4 Isokinetic dynamometer. This test involves extraordinarily low injury risk and is commonly used in rehabilitation settings in order to get a scientific measurement of leg strength.
Are there any risks or discomforts? The risks associated with participating in this study are: 1) The chance of a hematoma (bruise) formation from the muscle biopsy procedure, fat biopsy procedure or venipuncture.
2) Mild discomfort (i.e., pressure) from the muscle or fat biopsy procedures or venipuncture.
3) There is a rare chance that muscle or fat biopsy can lead to dull pain beyond the day of testing, become infected, or produce non-stop bleeding. To this end, hundreds of studies have been performing this procedure, and many of these studies have been performed on non-medical campuses. Furthermore, Drs. Mike Roberts and Dr. David Pascoe are experts of this procedure, and have performed hundreds using sterile techniques without any complications. It should be also noted that Highstead et al. (J Appl Physiol. 2005 Apr;98(4):1202-6) reported that only 18/1,301 participants experienced a hematoma after collection, only 2/1,301 participants experienced a persistent bleeding/oozing, only 4/1,301 participants experienced pain longer than 3 days after collection, and 0/1,301 participants experienced infection. Hence, these biopsy procedures present minimal risk to the participant when done using sterile technique. Also, please be upfront on the medical history questionnaire about being on blood-thinning medications and verbally let an investigator know that you are on them. 4) Mild discomfort (i.e., muscle soreness) from resistance training. Delayed onset muscle soreness is a likely consequence of exercise training and is not unusual. The muscle soreness is not considered a risk, but a nuisance that resolves itself over time.
5) The BIODEX machine is a form of exercise testing. There is an extraordinarily low risk that an adverse event can occur during exercise testing. To this end, and according to the 2009 ACSM Guidelines for Exercise Testing and Prescription, the risk of death associated with exercise testing is 0.05 percent of 10,000 people in a healthy population.
6) There is the risk that an allergic reaction may occur to lidocaine, betadine or ultrasound gel use. To reduce this risk, we will ask that you are upfront about all allergies in the medical history questionnaires and verbally tell the investigators any allergies that you may have. Are there any benefits to yourself or others?
You may increase your strength and muscle mass with training and/or supplementation. You may also enjoy the protein supplements as they are highly palatable.
This study will benefit the scientific community and sports performance practitioners by obtaining data which supports or refutes protein supplementation (with different protein sources) with the intent of building muscle mass and increasing strength.
Will you receive compensation for participating? To thank you for your time you will receive $500 upon completion of the study.
Between visits 4 thru 38, if you miss more than 3 whole body workouts (which would equal less than a 90% compliance rate to the protocol) then you will be disqualified from the study.
Partial compensation for the study are as follows: • $50 for completion of visits 1-2 (1 fat biopsy and 1 muscle biopsy given) • An additional $100 for completion of visits 1-3 (1 fat biopsy and 2 muscle biopsies given) • An additional $200 for completion of up to all visits except visit #39 (meaning that all workouts were completed but the last testing session was missed and, thus, the last muscle and fat biopsy was not taken) • An additional $150 for completion of visit #39 Please be aware that compensation for participation in research may be subject to taxation. If you have any questions regarding taxation, please contact the Office of Procurement and Payment Services (334-844-7771).
Are there any costs for participating? If you decide to participate, you will not be monetarily charged for anything.
In the unlikely event that you sustain an injury from participation in this study, the investigators have no current plans to provide funds for any medical expenses or other costs you may incur. If you change your mind about participating, you can withdraw at any time during the study. Your participation is completely voluntary. If you choose to withdraw, your data can be withdrawn as long as it is identifiable. Your decision about whether or not to participate or to stop participating will not jeopardize your future relations with Auburn University or the School of Kinesiology.
Your privacy will be protected. Any information obtained in connection with this study will remain confidential. Information obtained through your participation may be published in a professional journal and presented at a professional scientific meeting. However, you will be assigned a participant code upon agreeing to partake in the study and any discussion regarding your data will be associated with your participant code. Only de-identified data will be used to disseminate scientific results, and no one will use this data for commercial use (i.e., marketing). Given that this document contains sensitive personal health information it will be retained behind two locked doors in the office of the Principal Investigator (PI, Michael Roberts, PhD) and will only be accessed by the PI or involved study personnel.

If you have questions about this study, please ask them now or contact:
Finally, data collected on these forms will not be disclosed to a third party.
The sole use of this form is to further screen you for study inclusion/exclusion criteria. If there is further question as to your ability to participate in this study, the PI will consult with Dr. Trent Wilson and/or Dr. Michael Goodlett, MD. In this situation, the PI will discuss the case using your participant number and not your name. One year after the study concludes this document will be destroyed. Whole-body workout (3 sets of 6 reps is the goal, 2 min between sets) Squat Set 1: ______ lbs x _______ reps Set 2: ______ lbs x _______ reps Set 3: ______ lbs x _______ reps Bench press Set 1: ______ lbs x _______ reps Set 2: ______ lbs x _______ reps Set 3: ______ lbs x _______ reps Deadlift Set 1: ______ lbs x _______ reps Set 2: ______ lbs x _______ reps Set 3: ______ lbs x _______ reps Row Set 1: ______ lbs x _______ reps Set 2: ______ lbs x _______ reps Set 3: ______ lbs x _______ reps *crunches

Memo for: IRB protocol # 15-320 MR 1508 (modification request) "The Effects of Different Protein and Amino Acid Supplements on Muscle Hypertrophy after 12 Weeks of Resistance Exercise in Untrained Men"
Dear IRB Administration, As usual, we really appreciate the time and effort put into this review. The lab and students also consistently comment 'how lucky we are to be at Auburn where we can do cool human research'. This is very important for three reasons: 1) I believe the aforementioned sentiment is chiefly due to you all facilitating research rather than being a hindrance so this is much appreciated by myself and the students.
2) The human research approved by you all is giving all of the PhD student GREAT career training and making them competitive for post-Auburn positions (i.e., professorships or post-docs).
3) The human research the is being published is garnering great respect in the field of sports nutrition and Auburn Kinesiology is beginning to be viewed as a hub of quality research in this area from other academic institutions and industry sponsors.
With regard to the current protocol, we have addressed each area of concern in a point-by point fashion.
If we have not adequately provided details needed, do not hesitate to contact me.
Finally, it should be noted that the first set of files in this document are all highlighted with changes made. These pages are followed by clean versions of these documents for stamping/approval purposes.
Thank you again and I look forward to our future correspondence. The AU IRB's comments are as follows: 1. # 9 Rationale for Each Change: a. Amendment Request #2 -"Bent-over rows" is listed twice, while "deadlift", which is depicted on the next page, is not mentioned at all. Please correct.
-I apologize for this. This has been changed.
b. The "full body workout" needs to be consistent throughout the informed consent, protocol, and data sheets.
-Where applicable, I have added 'whole body workout to the consent form, protocol, and data collection sheet.
c. Amendment Request #3 -In this section, it is stated that the fat biopsy will be taken from the "upper buttock region," but in the informed consent on page 11, it is said that it would be taken from the "left leg". Also on page 6 of the informed consent, it was stated that the fat biopsy would be taken from the "hip." Please make the location of fat biopsy consistent in all parts of the protocol and informed consent.
-I apologize for this inconsistency. It is the upper buttock region and this has been changed throughout.
d. Amendment Request #4 -Please include another bullet that states that an extra $150 will be given when the final biopsies are completed.
-This now reads: "• $50 for completion of visits 1-2 (1 fat biopsy and 1 muscle biopsy given) • An additional $100 for completion of visits 1-3 (1 fat biopsy and 2 muscle biopsies given) • An additional $200 for completion of up to all visits except visit #39 (meaning that all workouts were completed but the last testing session was missed and, thus, the last muscle and fat biopsy was not taken) • An additional $150 for completion of visit #39 If this is not what you're aiming towards I can make further changes."
-I apologize for this inconsistency. This has been changed.
c. Risks or Discomforts: i. #1 should also include the fat biopsy as a procedure that could cause a hematoma.
-I apologize for this omission. This has been added.
ii. #6 should also include the ultrasound gel as a possible allergen for participants.
-I apologize for this omission. This has been added. iii. Include a question that asks whether the participant is an employee or a foreign national.
At the end of the IRB I have placed the following: "For payment purposes please indicate here if you are a: __________ employee __________ foreign national In both situations, taxes will be deducted prior to payment being received." e. Costs for participating: i. Change the current sentence to read: "In the unlikely event that you sustain an injury from participation in this study, the investigators have no current plans to provide funds for any medical expenses or other costs you may incur." -I have made this change.

Script and Advertisement:
a. Define what BCAAs are. b. Define location of fat biopsy throughout (as mentioned above).
-I have made these changes.

Data sheets:
a. Make the "full body workout" components consistent throughout.