The purpose of this study was to determine whether hypohydration reduces skeletal muscle endurance and whether increased H+ and Pi might contribute to performance degradation. Ten physically active volunteers (age 21-40 yr) performed supine single-leg, knee-extension exercise to exhaustion in a 1.5-T whole body magnetic resonance spectroscopy (MRS) system when euhydrated and when hypohydrated (4% body wt). 31P spectra were collected at a rate of one per second at rest, exercise, and recovery, and were grouped and averaged to represent 10-s intervals. The desired hydration level was achieved by having the subjects perform 2-3 h of exercise in a warm room (40 degrees C dry bulb, 20% relative humidity) with or without fluid replacement 3-8 h before the experiment. Time to fatigue was reduced (P < 0.05) by 15% when the subjects were hypohydrated [213 +/- 12 vs. 251 +/- 15 (SE) s]. Muscle strength was generally not affected by hypohydration. Muscle pH and Pi/beta-ATP ratio were similar during exercise and at exhaustion, regardless of hydration state. The time constants for phosphocreatine recovery were also similar between trials. In summary, moderate hypohydration reduces muscle endurance, and neither H+ nor Pi concentration appears to be related to these reductions.