Arm sweat was collected from nine male and nine female athletes sitting in a hot environment (HR, 40°C) and exercising at 50% ˙VO2max in neutral (NE, 25°C) and hot (HE, 35°C) environments to compare sweat iron concentrations and losses. Whole body sweat rate was determined from weight loss at 30-min intervals for 1 h. Sweat iron concentration was significantly higher during HR and NE than during HE, but was not significantly different between males and females. During exercise, sweat iron concentration decreased significantly from 30 to 60 min. Whole body sweat rates differed significantly across all environments with the highest rate in HE and the lowest in HR. Males lost significantly more sweat during exercise than females, but the difference at rest was not significant. Estimated whole body iron loss was significantly greater during exercise (NE = 0.08 mg·m-2·h-1, HE = 0.077 mg·m-2·h-1) than rest (0.039 mg·m-2·h-1), and the average iron loss was significantly greater in males (0.09 mg·m-2·h-1) than females (0.04 mg·m-2·h-1). Results suggest that the use of resting sweat iron concentration to estimate iron loss for 1 h of exercise in neutral environments is accurate but may overestimate iron loss during exercise in hot environments and during more prolonged exercise.