Muscle-damaging exercise (e.g., downhill running [DHR]) or heat exposure bouts potentially reduce physiological and/or cellular stress during future exertional heat exposure; however, the true extent of their combined preconditioning effects is unknown. Therefore, this study investigated the effect of muscle-damaging exercise in the heat on reducing physiological and cellular stress during future exertional heat exposure. Ten healthy males (mean±Standard Definition; age, 23±3years; body mass, 78.7±11.5kg; height, 176.9±4.7cm) completed this study. Participants were randomly assigned into two preconditioning groups: (a) DHR in the heat (ambient temperature [Tamb], 35°C; relative humidity [RH], 40%)and (b) DHR in thermoneutral (Tamb, 20°C; RH, 20%). Seven days following DHR, participants performed a 45-minflat run in the heat (FlatHEAT [Tamb, 35°C; RH, 40%]). During exercise, heart rate and rectal temperature (Trec) were recorded at baseline and every 5-min. Peripheral blood mononuclear cells were isolated to assess heat shock protein 72 (Hsp72) concentration between conditions at baseline, immediately post-DHR, and immediately pre-FlatHEAT and post-FlatHEAT. Mean Trec during FlatHEAT between hot (38.23±0.38°C) and thermoneutral DHR (38.26±0.38°C) was not significantly different (P=0.68), with no mean heart rate differences during FlatHEAT between hot (172±15beatsmin-1) and thermoneutral conditions (174±8beatsmin-1; P=0.58). Hsp72 concentration change from baseline to immediately pre-FlatHEAT was significantly lower in hot (-51.4%) compared to thermoneutral (+24.2%; P=0.025) DHR, with Hsp72 change from baseline to immediately post-FlatHEAT also lower in hot (-52.6%) compared to thermoneutral conditions (+26.3%; P=0.047). A bout of muscle-damaging exercise in the heat reduces cellular stress levels prior to and immediately following future exertional heat exposure.