Thirty apparently healthy males with a mean age of 20.43 ± 1.71 years, height of 176.67 ± 8.02 cm, and total body mass of 80.35 ± 18.52 kg served as participants in the study. The participants were not resistance-trained [not following a consistent resistance training program (i.e. thrice weekly) for at least one year prior to the study], but were recreationally-active. All participants were cleared for participation by passing a mandatory medical screening. Participants with contraindications to exercise as outlined by the American College of Sports Medicine and/or who had consumed any nutritional supplements (excluding multi-vitamins) such creatine monohydrate or various androstenedione derivatives or pharmacologic agents such as anabolic steroids three months prior to the study were not allowed to participate. All eligible subjects signed a university-approved informed consent document. Additionally, all experimental procedures involved in this study conformed to the ethical considerations of the Helsinki Code.
The study included baseline testing at day 0, followed by testing sessions at days 6, 27, and 48 in which blood and muscle samples were obtained and where body composition and muscle performance tests were performed.
The leg press and bench press maximal strength tests (Nebula, Versailles, OH) were performed by the participants to measure any changes in muscular strength during the course of the study. Four one repetition maximum (1-RM) strength tests were performed during the study at days 0, 6, 27, and 48. Initially, an estimated 50% (1-RM) measured from the previous testing 1-RM test, was utilized to complete 5 to 10 repetitions. After a two minute rest period, a load of 70% of estimated (1-RM) was utilized to perform 3 to 5 repetitions. Weight was gradually increased until a 1-RM was reached with each following lift, with a two-minute rest period in between each successful lift. Test-retest reliability of performing these strength assessments on subjects within our laboratory has demonstrated low mean coefficients of variation and high reliability for the bench press (1.9%, intraclass r = 0.94) and leg press (0.7%, intraclass r = 0.91), respectively.
Anaerobic power test
Anaerobic power was determined during each of the four testing sessions at days 0, 6, 27, and 48, and expressed relative to body mass. The determinations were made by performing a 30-second Wingate test on a computerized Lode cycle ergometer (Groningen, Netherlands). A warm-up of 30 rpm for 120 seconds was followed by maximal sprint for 30 seconds against a workload of 0.075 kg/kg of body weight. Correlation coefficients of test-retest reliability of performing these assessments of absolute peak power and mean power on participants within our laboratory has been found to be r = 0.692 and r = 0.950, respectively.
Body composition assessment
Total body mass (kg) was determined on a standard dual beam balance scale (Detecto Bridgeview, IL). Percent body fat, fat mass, and fat-free mass were determined using DEXA (Hologic Discovery Series W, Waltham, MA). Quality control calibration procedures were performed on a spine phantom (Hologic X-CALIBER Model DPA/QDR-1 anthropometric spine phantom) and a density step calibration phantom prior to each testing session. The DEXA scans were segmented into regions (right & left arm, right & left leg, and trunk). Each of these segments was analyzed for fat mass, lean mass, and bone mass. A sub-region was utilized to determine right thigh mass. The isolated region extended medially to the pubic symphysis down to the head of the femur. Total body water and compartment-specific fluid volumes were determined by bioelectric impedance analysis (Xitron Technologies Inc., San Diego, CA) using a low energy, high frequency current (500 micro-amps at a frequency of 50 kHz). Based on previous studies in our laboratory, the accuracy of the DEXA for body composition assessment is ± 2% as assessed by direct comparison with hydrodensitometry and scale weight.
Participants were randomly assigned to one of three groups in a double blind manner in which they orally ingested capsules and powder which contained either dextrose placebo [PLC (AST Sport Science, Colorado Springs, CO)], creatine monohydrate [CRT (Integrity Nutraceuticals, Sarasota, FL)], or creatine ethyl ester [CEE (Labrada Nutritionals, Houston, TX)]. For CRT, each capsule contained 250 mg of creatine monohydrate; however, for CEE each capsule contained 700 mg of creatine ethyl ester. Quality control testing of the creatine ethyl ester supplement using NMR from an independent laboratory from the University of Nebraska determined the product to contain 100% creatine ethyl ester HCL, with no detectable creatine HCL or creatinine HCL. The creatine supplement was shown to contain 99.8% creatine monohydrate and 0.2% creatinine.
After baseline testing procedures and fat-free mass determination by DEXA, supplements placebo were ingested relative to fat-free mass based on previous guidelines  for 48 days (loading from days 1–5 and maintenance from days 6–48.). Specifically, supplements were ingested at a relative daily dose of 0.30 g/kg fat-free body mass (approximately 20 g/day) during the loading phase, and at a relative daily dose of 0.075 g/kg fat free mass (approximately 5 g/day) during the maintenance phase. After the initial baseline assessment of body composition at day 0, supplement dosages were subsequently adjusted based on body composition assessments performed at days 6 and 27.
In order to standardize supplement intake throughout the study, participants were instructed to ingest the supplements in two equal intervals, one in the morning and one in the evening, throughout the day during the loading phase , and at one constant interval, in the morning, during the maintenance phase. Compliance to the supplementation protocol was monitored by supplement logs and verbal confirmation. After completing the compliance procedures the subjects were given the required supplement dosage for the following supplementation period.
Resistance training protocol
Participants engaged in a 4-day per week resistance-training program split into two upper and two lower extremity workouts per week for a total of seven weeks. The upper body resistance-training program consisted of nine exercises (bench press, lat pull, shoulder press, seated rows, shoulder shrugs, chest flies, biceps curl, triceps press down, and abdominal curls) twice per week and a seven exercise lower extremity program (leg press or squat, back extension, step ups, leg curls, leg extension, heel raises, and abdominal crunches) performed twice per week. We have previously shown this program to be effective at promoting significant gains in muscle strength and mass . Participants performed 3 sets of 8–10 repetitions with 70–80% 1-RM. Rest periods between exercises lasted no longer than three minutes and rest between sets lasted no longer than two minutes. Training sessions were not supervised, but were documented in training logs, and signed off to verify compliance and to monitor progress.
Muscle biopsies and venous blood sampling
Based on our previously-established guidelines , at each of the four testing sessions at days 0, 6, 27, and 48 percutaneous muscle biopsies (50–70 mg) were obtained using a Bergstrom (5 mm) needle. Muscle samples were obtained from the middle portion of the vastus lateralis muscle of the dominant leg at the midpoint between the patella and the greater trochanter of the femur, at a depth between one and two cm. For the remaining three biopsies, attempts were made to extract tissue from approximately the same location as the initial biopsy by using the pre-biopsy scar, depth markings on the needle, and a successive incision that was made approximately 0.5 cm to the former from medial to lateral. After removal, the muscle specimens were immediately frozen in liquid nitrogen and then stored at -80°C for later analysis.
At each of the four testing sessions, venous blood samples were obtained from the antecubital vein using a standard Vacutainer apparatus. Once collected, the samples were centrifuged for 15 minutes. The serum was removed and frozen at -80°C for later analysis. An 8-hour fast prior to blood donation was required for the participants before each of the four testing sessions.
Muscle and serum creatine analysis
Muscle tissue samples were analyzed spectrophotometrically for total creatine by the diacetyl/α-napthtol reaction . Using similar methods, serum samples were measured in duplicate for creatine concentration. Serum samples were immediately ready for creatine analysis, whereas muscle tissue had to first be prepared. For serum creatine analysis, duplicates for all samples yielded a coefficient of variation of 5.4%.
Approximately 10–15 mg of muscle tissue was cut and placed in a microfuge tube, and then placed in a vacuum centrifuge (Savant ISS110 SpeedVac™ Concentrator, Thermo Scientific, Milford, MA) to be spun for 18–24 hours. After sufficient muscle drying, the samples were then placed in an ultra-low freezer at -80°C. Dried muscle was powdered by grinding on a porcelain plate with a pestle. Connective tissue was removed and discarded, whereas powdered muscle was placed into pre-weighed microfuge tubes. Powdered muscle was extracted in a 0.5 M perchloric acid/1 mM EDTA solution on ice for 15-minutes, while periodically vortexing. Samples were then spun at 15,000 rpm at 4°C for 5-minutes. The supernatant was transferred into a microfuge tube and neutralized with 2.1 M KHCO3/0.3 M MOPS solution and then centrifuged again at 15,000 rpm for 5-minutes. In order to determine muscle total creatine concentration, supernatant from the above reaction was combined with ddH2O and 0.4 N HCl and heated at 65°C for 10-minutes to hydrolyze phosphate groups. The solution was then neutralized with of 2.0 N NaOH and the samples were allowed to incubate at room temperature allowing for color formation, which was detected by a spectrophotometer at 520 nm. Then the samples were run in duplicate against a standard curve of known creatine concentrations. The mean correlation coefficient of variation between duplicates was 1.53%. The standard curve correlation coefficient between plates for total muscle creatine was 0.998.
Dietary intake records and supplementation compliance
Throughout the course of the study, participants' dietary intake was not supervised; however, it was required that all participants keep detailed dietary records and not change their routine dietary habits throughout the course of the study. As such, participants were required to keep weekly physical activity records and four-day dietary records (three weekdays and one weekend) prior to each of the four testing sessions. The four-day dietary recalls were evaluated with the Food Processor dietary assessment software program (ESHA Research, Salem, OR) to determine the average daily macronutrient consumption of fat, carbohydrate, and protein. The participants were instructed to turn in their dietary records during each testing session. Each participant returned all of their dietary evaluations at the required time points for a 100% compliance rate. In an effort to ensure compliance to the supplementation protocol, participants were supplied with the appropriate amount of supplement to be ingested during the time between last three testing sessions. Upon reporting to the lab for each testing session at days 6, 27, and 48, participants returned the empty containers they had acquired between testing sessions
Reported side effects from supplements
At the last three testing sessions, participants reported by questionnaire whether they tolerated the supplement, supplementation protocol, as well as report any medical problems/symptoms they may have encountered throughout the study.
Data were analyzed using separate 3 (group) × time  univariate analysis of variance (ANOVA) with repeated measures on the time factor with SPSS for Windows Version 16.0 software (SPSS inc., Chicago, IL). Significant differences among groups were identified by a Tukey HSD post-hoc test. A probability level of ≤ 0.05 was adopted throughout.