The tubing terminated at a two-way valve which

opened and closed the Douglas bag. A known volume (range between 200–350 ml/min) of expired air was extracted through the sampling port of the Douglas bag at a constant flow rate, controlled by a flow meter. This air passed into a gas analyzer (Servomex Birinapant nmr 1440 Gas Analyzer, Servomax Group Limited, East Sussex, England) to determine the percentage of oxygen (O2) and carbon dioxide (CO2). The remaining volume of expired air in each Douglas bag was measured by evacuation through a dry gas meter (Harvard Apparatus Inc, Holliston, USA). The temperature of the air in Douglas bag was measured during evacuation. The gas analyzer was calibrated before each sample analysis with nitrogen, a calibration gas (BOC Gases, BOC limited, Surrey, UK). Barometric pressure was recorded. The measured expired gas volumes were

corrected to standard temperature and pressure for a dry gas using the universal gas equation. Inspired gas volume was derived using the Haldane transformation and used to calculate O2 and CO2, and RER as CO2/O2. Following the 40 min constant load exercise, the resistance was decreased to 10 W and participants were instructed to continue pedaling for an additional minute. The participant then commenced the 16.1 km (10 mile) self-paced time trial BMN 673 research buy on the same cycle ergometer used in the constant load phase. Nude BM was measured post exercise and the difference before and after completion of exercise was used to estimate sweat loss and sweat rate. The time to completion of the time trial was recorder but only revealed to the participants upon completion 5-Fluoracil of all trials. Blood treatment and analysis In all trials, blood was drawn into dry syringes and 8 mL dispensed into two 4 mL tubes containing K3EDTA while the remaining 2 mL were

dispensed into plain tubes. Duplicate aliquots (100 μL) of whole blood from the K3EDTA tube were rapidly deproteinized in 1000 μL of ice-cold 0.3-mol/L perchloric acid, centrifuged, and the supernatant used to measure Glu and lactate using standard enzymatic methods with spectrophotometer detection (Spectra Max M2 microplate reader). The remaining blood from the K3EDTA tube was analyzed for haemoglobin (cyanmethemoglobin method, Sigma, Chemical Company Ltd., Dorset, UK) and packed cell volume (conventional michrohematocrit method). The blood in the tube without anticoagulant was allowed to coagulate and then was centrifuged (8 min, 14,000 rpm, RT, Hettich Mikro 120); serum was collected and used to measure osmolarity by freezing point depression (Micro-osmometer 3300, Vitech Scientific, West Sussex, UK).