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 Gemcitabine cell line 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; JNK inhibitor screening library however, it was required that all participants keep detailed dietary records and not for 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.