e., 20–21°C and 30-40% relative humidity). The LT was estimated as work load at which the break-point in the relationship between CO2 output (CO2) and oxygen consumption (O2) occurred and the ventilatory equivalent (E) for O2 (E/O2) started to increase systematically without a concomitant increase in the ventilator
equivalent for CO2 (E/CO2) [12]. During this test, participants cycled for 5 min at 20 W as a warm up with a gradual increment of 15 W/min thereafter until cadence could no longer be maintained above 50 revolutions/min. Respired volumes and gas concentrations were measured every 15 s using a metabolic cart (Quark CPET b2, Italy, Cosmed). Respired volumes were calibrated with a 3-L Smad inhibitor syringe using a range of different flow profiles (Hans Rudolph, Kansas City, MO) while respired gas concentrations were calibrated against precision-analyzed gas mixtures. Following the maximal incremental exercise test, participants reported to the laboratory on three separate occasions (i.e., at least one familiarization trial and two experimental trials). On all occasions, participants were required to
cycle for 40 min at a constant pre-determined work rate Selleck MG132 followed by a 16.1 km self paced time trial at 30°C and 70% relative humidity. On the first occasion, participants underwent a familiarization trial, in order to become familiar with CBL-0137 cell line the exercise protocol and experimental procedures. The work rate (WR) at which participants Pyruvate dehydrogenase lipoamide kinase isozyme 1 would exercise was calculated by adding 20% of the difference between the WR at the O2max and the WR at the LT. In cases when during familiarization trial the desired duration (i.e., 40 min constant load plus 16.1 km time trial) could not be achieved, the WR was decreased to WR at LT for subsequent trials. Prior to the actual experimental trials, familiarization trials
were completed until the variability of O2 of two consecutive trials was within 5% difference. No subject had to complete a third familiarization trial to achieve less than 5% variability. Following the familiarization trial, participants were matched for body mass (BM) and were randomized in a double-blind fashion to receive Cr/Gly/Glu or Cr/Gly/Glu/Ala. Participants were separated into two groups because of the long washout period associated with Cr [13]. Participants of the the Cr/Gly/Glu group were instructed to ingest 20 g/day (4 × 5 g/day) of Cr monohydrate (Creapure Creatine Monohydrate, Reflex Nutrition Ltd, UK), 2 g.kg-1 BM per day (4 × 0.5 g .kg-1 BM per day) of Gly (Glycerin, Care plus, Huddersfield, UK) and 150 g/day (4 × 37.