Exertional heatstroke (EHS) is an emergency with a high mortality rate, characterized by central nervous system dysfunctions. This study aims to establish a Heat acclimation/acclimatization (HA) rat model in locomotion to recapitulate the physical state of human in severe environment of high temperature and humidity, and investigate the mechanism of organism protection in HA. (2) Methods: Wistar rats were exposed to 36 °C and ran 2 h/d for 21 days, acquired thermal tolerance test was conducted to assess the thermotolerance and exercise ability. Core temperature and consumption of water and food were observed. Expression of HSP70 and HSP90 of different tissues were determined by WB. Pathological structure of brain tissue was detected with HE staining. Proteomics was used to identify the differently expressed proteins in cerebral cortex of different groups. And key molecules were identified by RT-PCR and WB. (3) Results: HA rats displayed stronger thermotolerance and exercised ability on acquired thermal tolerance test. Brain water content of HA + EHS group reduced compared with EHS group. HE staining revealed slighter brain injuries of HA + EHS group than that of EHS. Proteomics focused on cell death-related pathways and key molecules Aquaporin 4 (AQP4) related to cell edema. Identification results showed HA increased AQP4, Bcl-xl, ratio of p-Akt/AKT and Bcl-xl/Bax, down-regulated Cleaved Caspase-3. (4) Conclusions: This HA model can ameliorate brain injury of EHS by reducing cerebral edema and cell apoptosis, offering experimental evidence for EHS prophylaxis.Copyright © 2023. Published by Elsevier B.V.