BACKGROUND: Intracellular skeletal muscle water is redistributed into the extracellular compartment during periods of dehydration, suggesting an associated decline in muscle volume. The purpose of this study was to evaluate skeletal muscle volume in active (knee extensors (KE)) and less active (biceps/triceps brachii, deltoid) musculature following dehydration induced by exercise in heat. METHODS: Twelve participants (seven men, five women) cycled in the heat under two conditions: (1) dehydration (DHYD) resulting in 3% and 5% losses of estimated total body water (ETBW), which was assessed by changes in body mass, and (2) fluid replacement (FR) where 3% and 5% losses of ETBW were counteracted by intermittent (20 to 30 min) fluid ingestion via a carbohydrate-electrolyte beverage. During both conditions, serum osmolality and skeletal muscle volume (assessed by magnetic resonance imaging) were measured at baseline and at the 3% and 5% ETBW loss measurement points. RESULTS: In DHYD, serum osmolality increased at 3% (p = 0.005) and 5% (p < 0.001) ETBW losses, while FR decreased serum osmolality at the 5% loss of ETBW time point (p = 0.009). In DHYD, KE muscle volume declined from 1,464 ± 446 ml to 1,406 ± 425 ml (3.9%, p < 0.001) at 3% ETBW loss and to 1,378 ± 421 ml (5.9%, p < 0.001) at 5% ETBW loss. The largest decline in KE volume in DYHD occurred in the mid-belly (31 ml, p = 0.001) and proximal (24 ml, p = 0.001) regions of the grouped vasti muscles. There were no changes in volume for the biceps/triceps (p = 0.35) or deltoid (p = 0.92) during DHYD. FR prevented the loss of KE muscle volume at 3% (1,430 ± 435 ml, p = 0.074) and 5% (1,431 ± 439 ml, p = 0.156) ETBW loss time points compared to baseline (1,445 ± 436 ml). CONCLUSIONS: Following exercise in the heat, the actively contracting muscles lost volume, while replacing lost fluids intermittently during exercise in heat prevented this decline. These results support the use of muscle volume as a marker of water loss.