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Heat acclimation induces adaptations that improve exercise tolerance in hot conditions. Here we report novel findings into the effects of ultra-marathon specific exercise load in increasing hot ambient conditions on indices of heat acclimation. Six male ultra-endurance runners completed a standard pre-acclimation protocol at 208C ambient temperature (Tamb), followed by a heat acclimation protocol consisting of six 2 h running exercise-heat exposures (EH) at 60% _ V O2max on a motorised treadmill in an environmental chamber. Three EH were performed at 308C Tamb, followed by another three EH at 358C Tamb. EH were separated by 48 h within Tamb and 72 h between Tamb. Nude body mass (NBM), blood and urine samples were collected pre-exercise; while NBM and urine were collected post-exercise. Rectal temperature (Tre), heart rate (HR), thermal comfort rating (TCR) and rating of perceived exertion were measured pre-exercise and monitored every 5 min during exercise. Water was provided ad libitum during exercise. Data were analysed using a repeated measures and one-way analysis of variance (ANOVA), with post hoc Tukey’s HSD. Significance was accepted as PB 0.05. Overall mean Tre was significantly lower during 308C EH3 and 358C EH3 compared with their respective EH1 (similar to 0.20 and similar to 0.238C, respectively; PB0.05). Similarly, overall mean HR was significantly lower during 308C EH3 and 358C EH3 compared with their respective EH1 (8 and 7 bpm respectively; PB0.05). A significant decrease in overall mean TCR was observed during 358C EH3, compared with 358C EH1 (PB 0.05). Significant increases in resting pre-exercise plasma volume (estimated from Hb and Hct) were observed by 308C EH3 (7.9%; PB 0.05). Thereafter, plasma volume remained above baseline throughout the experimental protocol. Two EH of 2 h at 60% _ V O2max at 308C Tamb was sufficient to initiate heat acclimation in all ultra-endurance runners. Further, heat acclimation responses occurred with increasing EH to 358C Tamb. Preventing exertional heat illnesses and optimising performance outcomes in ultra-endurance runners may occur with exposure to at least 2 h of exercise-heat stress on at least two occasions in the days leading up to multi-stage ultra-marathon competition in the heat.