Five trained men were studied during 2 h of cycling exercise at 67% peak oxygen uptake at 20-22°C to examine the effect of fluid ingestion on muscle metabolism. On one occasion, the subjects completed this exercise without fluid ingestion (NF) while on the other they ingested a volume of distilled deionized water that prevented loss of body mass (FR). No differences in oxygen uptake during exercise were observed between the two trials. Heart rate was lower (P < 0.01) throughout exercise when fluid was ingested, and rectal temperature after 2 h of exercise was lower (38.0 ± 0.2 and 38.6 ± 0.2°C for FR and NF, respectively; P < 0.01), as was muscle (vastus lateralis) temperature (38.5 ± 0.4 and 39.1 ± 0.5°C for FR and NF, respectively; P < 0.05). Resting muscle ATP, creatine phosphate, creatine, glycogen, and lactate levels were similar in the two trials, as were the postexercise ATP, creatine phosphate, and creatine levels. In contrast, muscle glycogen was higher (P < 0.05) and muscle lactate was lower (P < 0.05) after 2 h of exercise in FR compared with NF. Net muscle glycogen utilization during exercise was reduced by 16% when fluid was ingested (318 ± 46 and 380 ± 53 mmol/kg dry weight for FR and NF, respectively; P < 0.05). These results indicate that fluid ingestion reduces muscle glycogen use during prolonged exercise, which may account, in part, for the improved performance previously observed with fluid ingestion.