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Heat acclimation is the best strategy to improve performance in a hot environment. Many athletes seeking the benefits of heat acclimation lack access to a hot environment for exercise and, thus, rely on overdressing to simulate environmental heat stress. It is currently unknown whether this approach produces the requisite thermoregulatory strain necessary for heat acclimation in trained men and women.To compare physiological and cellular responses to exercise in a hot environment (HOT; 40°C, 30% RH) with minimal clothing (clo = 0.87) and in a temperate environment (CLO; 15°C, 50% RH) with overdressing (clo = 1.89) in both men and women.HR, rectal temperature (Tre), mean skin temperature (Tsk), sweating rate (SR), and extracellular heat shock protein (eHSP)72 were measured in 13 (7 males, 6 females) well-trained runners (V˙O2max: 58.7 ± 10.7 mL·kg−1·min−1) in response to ~60 min of treadmill running at 50%–60% V˙O2max in HOT and CLO.Tre increased in both conditions, but the increase was greater in HOT (ΔTre HOT: 2.6°C ± 0.1°C; CLO 2.0°C ± 0.1°C; P = 0.0003). SR was also higher in HOT (1.41 ± 0.1 L h−1; CLO: 1.16 ± 0.1 L·h−1; P = 0.0001). eHSP72 increased in HOT (% change: 59% ± 11%; P = 0.03) but not in CLO (6% ± 2%; P = 0.31). Mean Tsk and HR were not different between HOT and CLO in men but were higher in HOT for women.These data support the idea that overdressing during exercise in a temperate environment may produce the high Tre, Tsk, HR, and SR necessary for adaptation, but these responses do not match those in hot, dry environments. It is possible that greater exercise stimulus, warmer environment, or more clothing may be required to allow for a similar level of acclimation.