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Exercise alone or in combination with environmental heat stress can elevate blood S-100β protein concentrations. However, the explanatory power of exercise with marked environmental heat stress on the appearance of S-100β is questionable. It is possible that the process of heat acclimation might afford additional insight.Determine the S-100β response to moderate-intensity exercise with heat strain before and after heat acclimation.Nine healthy male volunteers completed 10 consecutive days of heat acclimation consisting of up to 100 min of treadmill walking (1.56 m·s−1, 4% grade) in the heat (45°C, 20% relative humidity). Changes in HR, rectal temperature (Tre), and sweat rate (SR) were examined to determine successful acclimation. Area under the curve (AUC) for Tre greater than 38.5°C was calculated to assess cumulative hyperthermia. Blood samples were taken before and after exercise on days 1 and 10 and were analyzed for serum osmolality and S-100β concentration.All subjects displayed physiological adaptations to heat acclimation including a significant (P < 0.05) reduction in final HR (161 to 145 bpm) and Tre (39.0 to 38.4°C), as well as a modest (~10%) increase in SR (1.10 to 1.20 L·h−1; P = 0.09). No differences were observed in pre- to postexercise serum S-100β concentrations on day 1 or 10, and no differences were observed in S-100β values between days 1 and 10. No significant correlations were found between S-100β values and any variable of interest.S-100β concentrations do not necessarily increase in response to exercise-heat strain, and no effect of heat acclimation on S-100β could be observed despite other quantifiable physiological adaptations.