The purpose of this study was to examine the effects of a 2-hour exercise bout on sweat iron and zinc concentrations and losses in males and females. Nine male and 9 female recreational cyclists exercised at ~50% V̇O2peak in a temperate environment (Ta = 23 °C, RH = 51%). Sweat samples were collected for 15 min during each of four 30-min exercise bouts. No significant differences were observed between males’ and females’ sweat iron or zinc concentrations or losses. Sweat iron concentrations decreased significantly between 60 and 90 min of exercise. Sweating rates increased significantly from 30 to 60 min and remained constant during the second hour. Sweat iron losses were significantly lower during the second hour (0.042 mg/m2/h) than the first hour of exercise (0.060 mg/m2/h). Sweat zinc concentrations also decreased significantly over the 2-hour exercise bout. Dietary intakes of iron and zinc were not significantly correlated to sweat iron and zinc concentrations. Sweat iron and zinc losses during 2 hours of exercise represented 3% and 1% of the RDA for iron and 9% and 8% of the RDA for zinc for men and women, respectively. These results suggest a possible iron conservation that prevents excessive iron loss during prolonged exercise.