Using a simultaneous-dilution technique, we investigated body-fluid volume changes during exercise in seven males, during 50 min of cycling (50% maximal work rate) in hot (36.2 °C), temperate (22.0 °C) and cool conditions (14.4 °C). Total body water (TBW), extracellular fluid (ECFV), plasma (PV) and erythrocyte volumes (RCV) were measured, while blood volume (BV), interstitial fluid volume (IFV), extracellular water (ECW) and intracellular water volumes (ICW) were derived. During the initial 10 min of cycling, BV decreased in all environments (P = 0.01), primarily because of a PV reduction (P = 0.01), while IFV, ECFV and ICW were not significantly changed. By 30 min, BV recovered in the temperate and cool conditions, despite mass losses of 563 and 520 mL (respectively), but remained depleted in the hot condition (P = 0.01). The 50-min volume changes revealed that, throughout exercise, body-fluid losses appeared to be drawn primarily from the extracellular space, regardless of air temperature. In the hot condition, the PV change represented 63% of the TBW loss, with the ICW contributing 23%. It was concluded that, during cycling, progressive dehydration mainly affected the extracellular space, with the intravascular and intracellular spaces being defended in less stressful conditions.