Aim This experiment was designed to test the hypothesis that the plasma volume is not selectively defended during exercise‐ and heat‐induced dehydration following humid‐heat acclimation. Methods Eight physically active males were heat acclimated (39.8 °C, relative humidity 59.2%) using 17 days of controlled hyperthermia (core temperature: 38.5 °C). Inter‐compartmental fluid losses and movements were tracked (radioisotopes and Evans blue dye) during progressive dehydration (cycling) in these same conditions and also during a resting recovery without fluid replacement (28 °C), before (day 1), during (day 8) and after heat acclimation (day 22). Results On days 8 and 22, there were significant increases in total body water, interstitial fluid and plasma volume (P < 0.05), but the intracellular compartments did not change (P > 0.05). The baseline plasma volume remained expanded throughout: 43.4 [±2.6 (day 1)], 49.1 [±2.4 (day 8); P < 0.05] and 48.9 mL kg−1 [±3.0 (day 22); P < 0.05]. During progressive dehydration, plasma reductions of 9.0% (±0.9: day 1), 12.4% (±1.6: day 8) and 13.6% (±1.2: day 22) were observed, with day 8 and 22 losses significantly exceeding day 1 (P < 0.05). During recovery, plasma volume restoration commenced, with the intracellular fluid contribution becoming more pronounced as acclimation progressed. Conclusion It is concluded that the plasma volume was not defended more vigorously following humid‐heat acclimation. Indeed, a greater fluid loss may well underlie the mechanisms for enhancing plasma volume recovery when heat acclimation is induced using the controlled‐hyperthermia technique.