Objective To compare the effects of different exercise acclimatization (EA) durations on liver injury and inflammatory response in mice with exertional heatstroke (EHS). Methods A total of 168 male C57BL/6 mice were randomly assigned to four groups using a random number table: no exercise acclimation group (EA0W, n=54), 1-week exercise acclimation group (EA1W, n=54), 2-week exercise acclimation group (EA2W, n=54), and blank control group (n=6). The blank control group did not undergo acclimatization training or EHS modeling. The EA1W and EA2W groups underwent daily 2-hour exercise training at a speed of 10 m/min in an environment maintained at (26.0±0.5) ℃ for 1 and 2 weeks, respectively, followed by a 2-day rest after training completion. EHS modeling was performed in mice of EA0W, EA1W, and EA2W groups through running at 10 m/min under controlled environmental conditions (39.5 ℃ ambient temperature, 65% relative humidity). The modeling endpoint was defined as loss of consciousness accompanied by a core body temperature ≥42.7 ℃. All modeling procedures were systematically documented. Following modeling, 18 mice from EA0W, EA1W, and EA2W groups underwent 24-hour survival analysis. Blood samples from the abdominal aorta and liver tissues were collected at 6, 12 and 24 hours post-modeling (6 mice per time point for each group). Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) were quantified. Interleukin (IL)-1β and IL-6 concentrations were determined using enzyme-linked immunosorbent assay (ELISA). Liver tissue specimens underwent hematoxylin-eosin (HE) staining and pathological scoring. Results The EHS model was successfully established in all EA groups. When all mice in EA0W group developed EHS (65 min after the modeling initiation), the incidence rates in EA1W and EA2W groups were 50.0% and 22.2%, respectively, with a statistically significant difference between EA0W group and the latter two groups (P<0.05). When all mice in the three groups developed EHS, the time to EHS onset was significantly longer in both EA1W and EA2W groups compared to EA0W group, with EA2W group showing a longer onset time than EA1W group (P<0.05). Survival analysis revealed a significantly higher 24-hour survival rate in EA2W group (61.1%) compared to EA0W group (33.3%) (P<0.05), while no significant difference was observed between EA1W group and the other two groups (P>0.05). The levels of IL-1β, IL-6, and CK were highest at 6 h post-modeling in all EA groups (P<0.05), and liver injury was most severe at 12 h post-modeling (P<0.05). Compared to EA0W group, the levels of ALT, AST, and IL-1β, as well as liver pathology scores, were significantly lower at 12 h post-modeling in both EA1W and EA2W groups (P<0.05), with EA2W group showing significantly lower ALT and AST levels, as well as liver pathology scores than EA1W group (P<0.05). At 6 h post-modeling, CK levels were significantly higher in EA1W and EA2W groups compared to EA0W group (P<0.05), with EA2W group exhibiting higher CK levels than in EA1W group (P<0.05). Conclusions Exercise acclimation helps reduce the incidence of EHS. Following EHS onset, the survival rate of exercise-acclimated mice is higher than that non-acclimated mice, with a significantly higher survival rate in mice acclimated for 2 weeks compared to non-acclimated mice. However, no significant difference in survival rate is observed between mice acclimated for 1 week and non-acclimated mice. Additionally, exercise acclimation for 2 weeks is more effective in reducing liver injury and inflammatory responses compared to 1-week acclimation.