Studies in laboratory animals found that iron deficiency without anemia decreased oxidative capacity and increased reliance on carbohydrate as the substrate for energy, thereby causing impaired endurance. The purpose of this cross-sectional study was to investigate the relation between iron deficiency without anemia and physical performance in healthy active women aged 19-36 y. Iron-status assessment included determination of hemoglobin, hematocrit, transferrin saturation, and serum ferritin values. Dietary iron intake was assessed by frequency questionnaires and physical activity level was estimated by frequency questionnaires and 2-wk records. Fifteen women with normal iron status and 15 women with iron depletion (serum ferritin < 12 micrograms/L) were chosen randomly from a group of 69 nonanemic women and given physical-performance tests, including determinations of maximum oxygen consumption (VO2max), ventilatory threshold, and delta-efficiency. There were no significant differences between the two groups in body size, body composition, physical activity level, dietary iron intake, delta-efficiency, or ventilatory threshold. Compared with the iron-depleted group, the iron-sufficient group had significantly higher hemoglobin, transferrin saturation, and serum ferritin values and a significantly greater tendency to use iron supplements. When physical activity level and fat-free mass were controlled for, the iron-depleted group had a significantly lower VO2max. The difference in VO2max was significantly associated with serum ferritin concentration; hemoglobin value was not a significant confounder. Therefore, reduction of VO2max in nonanemic women with iron depletion was likely caused by factors related to reduced body iron storage but was unrelated to decreased oxygen-transport capacity of the blood.