Aims Dehydration, factors of hypvolemia and plasma hyperosmolality, attenuates heat tolerance in humans and mice. Themoregulation are achieved by autonomic and behavioral responses. However, we know a little about if and how the behavioral responses in heat, such as heat escape behavior or seeking cooler environment are activated by the exercise training. We evaluates the role of behavioral thermoregulation in heat tolerance. Methods All experimental procedures were conducted under the guidelines of the Japanese Physiological Society and Waseda University in Japan. Male ICR mice (6 w age) were housed with/without a running-wheel for 8 wks (WR and NWR groups, n=40 each). In the 5th week, under inhalation anesthesia with 1% isoflurane, they were placed a data-logger with temperature sensor for the measurement of body temperature (Tb) in the abdominal cavity. Mice were got familiarized with being in an experiment box (50 x 10 x 15 cm; made of Plexiglas with 5 temperature-controllable Peltier boards at the bottom), and learned either of following settings: 1) board temperature of 28°C or 39°C; 2) operant-behavior setting; each board was set at 39°C, and the right-end board was changed at 20°C within 60 sec only when a mice moved on the left two board; and 3) thermal mosaic setting; each board was set at either 15°C, 22°C, 28°C, 35°C, or 39°C with a 6-min interval. After the 8-wk training period, a mouse had subcutaneous injection of isotonic- or hypertonic-saline with 0.1 ml 0.5% lidocaine (1 ml/100 g of body wt; 154 or 2,500 mM, IS or HS subgroup), and was exposed to either of the setting for 2 h after 1.5 h baseline (Experiments 1 – 3). Results In Experiment 1, after the 39°C board exposure, Tb in both subgroups of the WR group was higher than that in the NWR group without any significant differences. In Experiment 2, the NWR group showed smaller operant counts in the HS subgroup than the IS subgroup; however, the WR group did not. In Experiment 3, the WR group had lower temperature of thermal preference than the NWR group without any differences between the subgroups (e.g. 33.4 ± 0.3°C and 34.7 ± 0.1°C in the IS subgroups of the WR and NWR groups, respectively). Conclusions Hyperosmotic condition was a stimulus decreasing heat tolerance in heat. However, exercise training in mice may have resulted in 1) lower thermal preference, and 2) greater activation of behavioral thermoregulatory response. Therefore, the training contributes to increase in the heat tolerance.