Our objective was to investigate the effects of iron depletion on adaptation to aerobic exercise, assessed by time to complete a 15-km cycle ergometer test. Forty-two iron-depleted (serum ferritin <16 microg/l), nonanemic (Hb >12 g/dl) women (18-33 yr old) received 100 mg of ferrous sulfate (S) or placebo (P) per day for 6 wk in a randomized, double-blind trial. Subjects trained for 30 min/day, 5 days/wk at 75-85% of maximum heart rate for the final 4 wk of the study. There were no group differences in baseline iron status or in 15-km time. Iron supplementation increased serum ferritin and decreased transferrin receptors in the S compared with the P group. The S and P groups decreased 15-km time and respiratory exchange ratio and increased work rate during the 15-km time trial after training. The decrease in 15-km time was greater in the S than in the P group (P = 0.04) and could be partially attributed to increases in serum ferritin and Hb. These results indicate that iron deficiency without anemia impairs favorable adaptation to aerobic exercise.