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BACKGROUND The protein klotho protects cellular function during various physiological stressors, such as exercise, however it is unknown how the age-related decline in klotho production affects responses during exercise, especially in the heat. OBJECTIVE Our objective was to determine the effect of exercise in temperate and hot environmental conditions on serum klotho concentrations in young and older men. METHODS 12 young (mean ± SD: 22 ± 3 years) and 12 older (59 ± 4 years) men performed 180 minutes of moderate-intensity treadmill walking (metabolic rate: 200 W/m2) in a temperate [wet-bulb globe temperature (WBGT) 16°C, achieved with 21.9°C, 35% relative humidity (RH)] and hot (WBGT 32°C, achieved with 41.4°C, 35% RH) environment. Serum klotho was assessed before and after exercise, as well as 60-min post-exercise recovery in the respective environments. RESULTS Absolute klotho concentrations were greater in young versus older men during all measured time points in the temperate (p = 0.032), but not the hot condition (p = 0.064). In the hot condition, the change in serum klotho from baseline was significantly higher after exercise in the heat (mean ± SEM: +251 ± 73 pg/mL) than the temperate (+75 ± 57 pg/mL) environment in both groups (p = 0.026). However, this elevation was not maintained during recovery. CONCLUSION We showed that prolonged exercise in a temperate environment does not elicit a klotho response in either group. In contrast, despite lower resting klotho levels, the older men showed a similar exercise-induced increase in serum klotho response as their younger counterparts.