The female sex hormone 17β-estradiol (E2) has been shown to increase lipid and decrease carbohydrate utilization in animals. We administrated oral E2 and placebo (randomized, double blind, crossover) to eight human male subjects for 8 days (∼3 mg/day) and measured respiratory variables, plasma substrates, hormones (E2, testosterone, leptin, cortisol, insulin, and catecholamines), and substrate utilization during 90 min of endurance exercise. [6,6-2H]glucose and [1,1,2,3,3-2H]glycerol tracers were used to calculate substrate flux. E2 administration increased serum E2 (0.22 to 2.44 nmol/l, P < 0.05) and decreased serum testosterone (19.4 to 11.5 nmol/l, P < 0.05) concentrations, yet there were no treatment effects on any of the other hormones. Glucose rates of appearance (Ra) and disappearance (Rd) were lower, and glycerol Ra-to-Rd ratio was not affected by E2 administration. O2 uptake, CO2production, and respiratory exchange ratio were not affected by E2; however, there was a decrease in heart rate ( P < 0.05). Plasma lactate and glycerol were unaffected by E2; however, glucose was significantly higher ( P < 0.05) during exercise after E2administration. We concluded that short-term oral E2administration decreased glucose Ra and Rd, maintained plasma glucose homeostasis, but had no effect on substrate oxidation during exercise in men.