After a warm-up period (depending on the runner), the subjects started running at 8 km·h-1 for 3 min in order to reach a steady state. In the next exercise bout the treadmill speed was set to 10 km·h-1 for 3 min and this procedure was repeated with 2 km·h-1 increments in running speed until volitional exhaustion of the subject. During the test expired gas samples (30 s collection 4EGI-1 in vivo time at the end of each bout) were taken using Douglas bag collection technique

as is considered the gold standard method [22] and analyzed for O2% and CO2% (Servopro 4100 Gas Purity Analyzer, Servomex, UK) as well as analyzed for volume using a dry gas meter (Harvard, Kent, UK) and temperature of expired gases. Barometric pressure was measured using a standard mercury barometer. Additionally, a HR monitor (Polar Sports Tester, Polar Electro Oy, Kempele, Finland) was attached prior to each test and HR was recorded at the end of each bout. The measurement

selleck chemicals was used for calculating the intensity (60% of ) that subjects would perform during the actual tests. Running speed at 60% of (exercise intensity) was calculated using the linear relation between treadmill speed and . Prior to the actual experimental trials, familiarization trials were completed until the variability of of two consecutive trials was within 5% difference. No subject had to complete a third familiarization trial to achieve less than 5% variability, an observation which is in line with our previous experience of trained runners [23]. At least three days after this familiarization period, subjects reported to the laboratory for the

first experimental trial (i.e., a pre-supplementation trial). After this baseline test, all subjects commenced the hyperhydration treatment comprising Cr, Gly and Glu. For this, subjects consumed a solution of 11.4 4��8C g of Cr·H2O (equivalent to 10 g Cr), (Reflex Creapure Creatine, Reflex Nutrition LTD, UK), 1 g·kg-1 of BM Gly (Glycerin BP/Value Health Glycerin BP, Boots Company plc) and 75 g of Glu polymer (SiS GO electrolyte), mixed in hot water (approximately 50°C) and made up in 1 L of cold water twice daily. This supplementation regimen was followed for 6 days. This protocol has been shown to increase resting muscle-phosphocreatine levels within 5 days [24]. On the day of the post-supplementation test (i.e., day 7th) subjects began check details consuming the final supplement 5 h before the exercise-performance trial (with instructions to complete ingestion within 1 h). Hypertonic solutions such as the Cr, Gly, Glu combination (~1556 mOsm·kg-1) cause an initial net secretion of water into the intestinal lumen [25], resulting in an effective loss of body water, albeit temporary.