Purpose To investigate the effects of 60 min daily, short-term (STHA) and medium-term (MTHA) isothermic heat acclimation (HA) on the physiological and perceptual responses to exercise heat stress. Methods Sixteen, ultra-endurance runners (female = 3) visited the laboratory on 13 occasions. A 45 min sub-maximal (40% W max) cycling heat stress test (HST) was completed in the heat (40 °C, 50% relative humidity) on the first (HST PRE), seventh (HST STHA) and thirteenth (HST MTHA) visit. Participants completed 5 consecutive days of a 60 min isothermic HA protocol (target T re 38.5 °C) between HST PRE and HST STHA and 5 more between HST STHA and HST MTHA. Heart rate (HR), rectal (T re), skin (T sk) and mean body temperature (T body), perceived exertion (RPE), thermal comfort (TC) and sensation (TS) were recorded every 5 min. During HSTs, cortisol was measured pre and post and expired air was collected at 15, 30 and 45 min. Results At rest, T re and T body were lower in HST STHA and HST MTHA compared to HST PRE, but resting HR was not different between trials. Mean exercising T re , T sk , T body , and HR were lower in both HST STHA and HST MTHA compared to HST PRE. There were no differences between HST STHA and HST MTHA. Perceptual measurements were lowered by HA and further reduced during HST MTHA. Conclusion A 60 min a day isothermic STHA was successful at reducing physiological and perceptual strain experienced when exercising in the heat; however, MTHA offered a more complete adaptation.