Metabolic effects of an overnight fast (postabsorptive state, PA) or a 3.5-day fast (fasted state, F) were compared in eight healthy young men at rest and during exercise to exhaustion at 45% maximum O2 uptake. Glucose rate of appearance (Ra) and disappearance (Rd) were calculated from plasma glucose enrichment during a primed, continuous infusion of [6,6-2H]glucose. Serum substrates and insulin levels were measured and glycogen content of the vastus lateralis was determined in biopsies taken before and after exercise. At rest, whole-body glucose flux (determined by the deuterated tracer) and carbohydrate oxidation (determined from respiratory exchange ratio) were lower in F than PA, but muscle glycogen levels were similar. During exercise, glucose flux, whole-body carbohydrate oxidation, and the rate of muscle glycogen utilization were significantly lower during the fast. In the PA state, glucose Ra and Rd increased together throughout exercise. However, in the F state Ra exceeded Rd during the 1st h of exercise, causing an increase in plasma glucose to levels similar to those of the PA state. The increase in glucose flux was markedly less throughout F exercise. Lower carbohydrate utilization in the F state was accompanied by higher circulating fatty acids and ketone bodies, lower plasma insulin levels, and the maintenance of physical performance reflected by similar time to exhaustion.