We studied if dehydrating exercise would reduce muscle water (H2Omuscle ) and affect muscle electrolyte concentrations. Vastus lateralis muscle biopsies were collected prior, immediately after, and 1 and 4 h after prolonged dehydrating exercise (150 min at 33 ± 1 °C, 25% ± 2% humidity) on nine endurance-trained cyclists (VO2max = 54.4 ± 1.05 mL/kg/min). Plasma volume (PV) changes and fluid shifts between compartments (Cl(-) method) were measured. Exercise dehydrated subjects 4.7% ± 0.3% of body mass by losing 2.75 ± 0.15 L of water and reducing PV 18.4% ± 1% below pre-exercise values (P < 0.05). Right after exercise H2Omuscle remained at pre-exercise values (i.e., 398 ± 6 mL/100 g dw muscle(-1)) but declined 13% ± 2% (342 ± 12 mL/100 g dw muscle(-1); P < 0.05) after 1 h of supine rest. At that time, PV recovered toward pre-exercise levels. The Cl(-) method corroborated the shift of fluid between extracellular and intracellular compartments. After 4 h of recovery, PV returned to pre-exercise values; however, H2Omuscle remained reduced at the same level. Muscle Na(+) and K(+) increased (P < 0.05) in response to the H2Omuscle reductions. Our findings suggest that active skeletal muscle does not show a net loss of H2O during prolonged dehydrating exercise. However, during the first hour of recovery H2Omuscle decreases seemly to restore PV and thus cardiovascular stability.