Objective To investigate the effects of active heat acclimatization training on plasma heat shock protein 70 (HSP70), cortisol, tetraiodothyronine (T4), and creatinine levels, and to identify effective evaluation factors for active heat acclimatization and establish an evaluation model. Methods A total of 170 healthy adult male volunteers were recruited and assigned to high or qualified endurance groups based on their 3-km running performance, and were further subdivided into heat acclimatization training group or control group, namely high endurance training (HET) group (n=60), high endurance control (HEC) group (n=30), qualified endurance training (QET) group (n=50), and qualified endurance control (QEC) group (n=30). The HET and QET groups underwent 7 and 12 d of outdoor active heat acclimatization training, respectively, primarily consisting of 5-km loaded fast walking. Plasma levels of HSP70, cortisol, T4, and creatinine were detected using enzyme-linked immunosorbent assay before and after training on the 1st day (baseline test) and the last day (heat acclimatization efficacy evaluation test). Multivariate logistic regression analysis was used to identify evaluation factors for active heat acclimatization, and a combined evaluation model was established. Receiver operating characteristic (ROC) curve was used to evaluate the evaluation performance of individual factors and their combination. Results After both the baseline test and the efficacy evaluation test, plasma levels of HSP70, cortisol, and creatinine in all subjects were significantly elevated compared with those before the baseline test (all P＜0.05). In the heat acclimatization training group, HSP70 and cortisol levels were significantly lower after the efficacy evaluation test than those after the baseline test (both P＜0.01), whereas creatinine level showed no significant change (P＞0.05). Although T4 level was significantly lower after the efficacy evaluation test than that after the baseline test (P＜0.01), there was no significant difference between the pre- and post-test of efficacy evaluation (P＞0.05). Both the HET and QET groups exhibited changes consistent with the overall training group. Multivariate logistic regression analysis indicated that plasma HSP70 and cortisol levels after the efficacy evaluation test could serve as evaluation factors for heat acclimatization, with area under curve (AUC) values of 0.836 and 0.791, sensitivities of 0.767 for both, and specificities of 0.809 and 0.718, respectively. The combination of both factors yielded an AUC of 0.887 in evaluating active heat acclimatization, with sensitivity and specificity of 0.882 and 0.750, respectively. The combined prediction model was logitP=7.466-0.303×HSP70-0.024×cortisol. Conclusion A single session of heat exposure training induces elevations in plasma HSP70 and cortisol. Active heat acclimatization reduces the magnitude of these elevations without altering resting levels, and these changes are not affected by differences in endurance capacity. Plasma HSP70 and cortisol can serve as evaluation factors for active heat acclimatization, and their combination may offer better predictive performance.