Most heat acclimation data are from regimes longer than 1 week, and acclimation advice is to prevent dehydration. Objectives: We hypothesized that (i) short-term (5-day) heat acclimation would substantially improve physiological strain and exercise tolerance under heat stress, and (ii) dehydration would provide a thermally independent stimulus for adaptation. Methods: Nine aerobically fit males heat acclimated using controlled-hyperthermia (rectal temperature 38.5°C) for 90 min on 5 days; once euhydrated (EUH) and once dehydrated (DEH) during acclimation bouts. Exercising heat stress tests (HSTs) were completed before and after acclimations (90-min cycling in Ta 35°C, 60% RH). Results: During acclimation bouts, [aldosterone]plasma rose more across DEH than EUH (95%CI for difference between regimes: 40–411 pg ml−1; P = 0.03; n = 5) and was positively related to plasma volume expansion (r = 0.65; P = 0.05), which tended to be larger in DEH (CI: −1 to 10%; P = 0.06; n = 9). In HSTs, resting forearm perfusion increased more in DEH (by 5.9 ml 100 tissue ml−1 min−1: −11.5 to −1.0; P = 0.04) and end-exercise cardiac frequency fell to a greater extent (by 11 b min−1: −1 to 22; P = 0.05). Hydration-related effects on other endocrine, cardiovascular, and psychophysical responses to HSTs were unclear. Rectal temperature was unchanged at rest but was 0.3°C lower at end exercise (P < 0.01; interaction: P = 0.52). Conclusions: Short-term (5-day) heat acclimation induced effective adaptations, some of which were more pronounced after fluid-regulatory strain from permissive dehydration, and not attributable to dehydration effects on body temperature. Am. J. Hum. Biol. 26:311–320, 2014. © 2014 Wiley Periodicals, Inc.