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 20°C ambient temperature (T amb), followed by a heat acclimation protocol consisting of six 2 h running exercise-heat exposures (EH) at 60% VO2max on a motorised treadmill in an environmental chamber. Three EH were performed at 30°C T amb, followed by another three EH at 35°C T amb. EH were separated by 48 h within T amb and 72 h between T amb. Nude body mass (NBM), blood and urine samples were collected pre-exercise, while NBM and urine were collected post-exercise. Rectal temperature (T re), 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 P< 0.05. Overall mean T re was significantly lower during 30°C EH3 and 35°C EH3 compared with their respective EH1 (-0.20 and-0.23°C, respectively, P<0.05). Similarly, overall mean HR was significantly lower during 30°C EH3 and 35°C EH3 compared with their respective EH1 (8 and 7 bpm respectively, P<0.05). A significant decrease in overall mean TCR was observed during 35°C EH3, compared with 35°C EH1 (P< 0.05). Significant increases in resting pre-exercise plasma volume (estimated from Hb and Hct) were observed by 30°C EH3 (7.9%, P< 0.05). Thereafter, plasma volume remained above baseline throughout the experimental protocol. Two EH of 2 h at 60% VO2max at 30°C T amb was sufficient to initiate heat acclimation in all ultra-endurance runners. Further, heat acclimation responses occurred with increasing EH to 35°C T amb. 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.