Klotho is a cytoprotective protein that increases during acute physiological stressors (e.g., exercise heat stress), although age-related declines in klotho may underlie cellular vulnerability to heat stress. The present study aimed to compare serum klotho in healthy older men and men with type 2 diabetes (T2D) or hypertension (HTN) during prolonged exercise in temperate or hot conditions. We evaluated serum klotho in 12 healthy older men (mean [SD]; 59years [4]), 10 men with HTN (60years [4]), and 9 men with T2D (60years [5]) before and after 180min of moderate-intensity (fixed metabolic rate of 200 W/m2; ~ 3.4 METs) exercise and 60min of recovery in temperate (wet-bulb globe temperature (WBGT) 16°C) and hot (WBGT 32°C) environments. Core temperature (rectal), heart rate (HR), and heart rate reserve (HRR) were measured continuously while klotho was measured at the end of baseline, exercise, and recovery. Total exercise duration was reduced during the hot condition in older men with HTN and T2D than healthy older men (both p ≤ 0.049), despite similar core temperatures, HR, and HRR. Klotho was higher than rest following exercise in the heat in healthy older men (+ 191pg/mL [189]; p < 0.001) and responses were greater (p = 0.036) than men with HTN (+ 118pg/mL [49]; p = 0.030), although klotho did not increase in men with T2D (+ 4pg/mL [71]; p ≥ 0.638). Given klotho's role in cytoprotection, older men with HTN and especially T2D may be at increased cellular vulnerability to prolonged exercise or physically demanding exercise in the heat.