To examine the effect of acute plasma volume expansion (PVE) on substrate selection during exercise, seven untrained men cycled for 40 min at 72 ± 2% peak oxygen uptake (O_{2 peak}) on two occasions. On one occasion, subjects had their plasma volume expanded by 12 ± 2% via an intravenous infusion of the plasma substitute Haemaccel, whereas on the other occasion no such infusion took place. Muscle samples were obtained before and immediately after exercise. In addition, heart rate and pulmonary gas and venous blood samples were obtained throughout exercise. No differences in oxygen uptake or heart rate during exercise were observed between trials, whereas respiratory exchange ratio, blood glucose, and lactate were unaffected by PVE. Muscle glycogen and lactate concentrations were not different either before or after exercise. In addition, there was no difference in total carbohydrate oxidation between trials (control: 108 ± 2 g; PVE group: 105 ± 2 g). Plasma catecholamine levels were not affected by PVE. These data indicate that substrate metabolism during submaximal exercise in untrained men is unaltered by acute hypervolemia.