Validation of a Heat Tolerance Test (HTT) to distinguish mice exposed to exertional heat stroke

The HTT was developed to determine heat tolerance in humans after an episode of exertional heat stroke (EHS). It can be useful in studies with rodents, because it requires a shorter heat-exposure time, thereby supporting both the rodent welfare and scientific validity. To validate an HTT as a tool to distinguish mice subjected to EHS from those exposed to other experimental stressors. We hypothesized that the HTT would be able to differentiate animals subjected to EHS from those exposed to chronic heat stress (CHS) or lipopolysaccharide (LPS) challenges. Twenty-eight female mice were randomly assigned to four groups: 1) Control (CON, n = 7); 2) CHS (n = 8); 3) LPS (n = 7); and 4) EHS (n = 6). One week after arrival at the animal facility, mice underwent laparotomy surgery for implantation of a core temperature sensor. After a 15-day recovery period, mice were subjected to a 30-day experimental protocol. The CON, LPS, and EHS groups were maintained at 24.5°C, while the CHS group was kept at 34.0°C. At the end of the 30-day protocol, after a 24-hour interval, the CON, CHS, and LPS groups were exposed to the HTT, during which animals remained at rest inside their home cages at 40°C for 40 minutes. Core temperature was recorded every 10 minutes. Three hours before the HTT, the LPS group received an intraperitoneal injection of lipopolysaccharide (100 µg/kg). Twenty-four hours after the 30-day protocol, the EHS group underwent a progressive exercise protocol in a hot environment (37.5°C) starting at a wheel speed of 2.5 m/min, with increments of 0.3 m/min every 10 minutes. Once core temperature reached 41°C, the speed was maintained until loss of consciousness. Twenty-four hours after the EHS protocol, these animals were subjected to the HTT. Two-way ANOVA (main factors: group x time) was used to compare core temperatures followed by Tukey’s post hoc test. Data are expressed as means ± standard deviation. During the HTT the EHS group exhibited significantly higher core temperatures compared with all other groups at minute 10 (EHS: 40.56 ± 0.25°C; CON: 39.80 ± 0.24°C; CHS: 39.59 ± 0.30°C; LPS: 39.48 ± 0.38°C; P < 0.001) and compared with the CON and CHS groups at minute 20 (EHS: 40.98 ± 0.33°C; CON: 40.40 ± 0.27°C; CHS: 40.28 ± 0.28°C; P < 0.05). From minute 30 onward, the LPS group displayed higher core temperatures relative to the CHS group at minutes 30 and 40 (minute 30: 40.90 ± 0.26°C vs. 40.41 ± 0.31°C, P = 0.0181; minute 40: 41.12 ± 0.30°C vs. 40.39 ± 0.27°C, P = 0.0009) and relative to the CON group at minute 40 (41.12 ± 0.30°C vs. 40.54 ± 0.25°C, P = 0.0064). At no time during the HTT were significant differences observed in the core temperatures between the CON and CHS groups. The HTT is an effective test for identifying mice previously exposed to EHS, with only 10 minutes of testing being sufficient to detect these differences. It is possible that the HTT is also an efficient tool for detecting other experimental stressors, as observed in the LPS group in the present study. Funding: DoD. USAMRAA. HT9425-23-PRMRP-FPA Grant log number BA220322 to OL. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.