To determine the effect of acute plasma volume (PV) expansion on substrate utilization, blood metabolites and catecholamines to prolonged, moderate intensity cycle exercise, eight untrained men mean maximal oxygen uptake VO2max 4.10 (SEM 0.32) 1.min-1 were infused (10 ml.kg-1) with a 6% dextran (DEX) solution. These responses were also compared to those elicited using a short-term training (TR) protocol involving cycling for 90 to 120 min.day-1 at 60% VO2max for 3 consecutive days. In general DEX, which resulted in a calculated expansion of PV by 23.9% was without effect in modifying exercise oxygen uptake or the reduction in the respiratory exchange ratio (R) observed during prolonged exercise. In addition, the concentrations of blood glucose, glycerol, alanine and serum free fatty acids, although altered (P < 0.05) by exercise, were not altered by DEX. Blood lactate concentration was only higher (P < 0.05) at 30 min of exercise during DEX compared to the control. With the exception of blood lactate concentration, which was reduced (P < 0.05), TR did not change R or the concentrations of other blood metabolites. The concentrations of nonadrenaline and adrenaline, were depressed (P < 0.05) by DEX and TR at 60 and 90 min of exercise. These results would suggest that mechanisms as yet undefined can compensate for the estimated 10% reduction in arterial oxygen content mediated by acute PV expansion and enable prolonged exercise to be performed without adjustments in substrate selection and substrate mobilization.