Reduced carbohydrate (CHO) availability after exercise has a potent influence on the regulation of substrate metabolism, but little is known about the impact of fat availability and/or energy deficit on fuel metabolism when dietary CHO availability is not reduced. The purpose of this study was to determine the influence of a post-exercise energy deficit, independent of CHO availability, on plasma substrate concentrations and substrate oxidation. Seven moderately-trained men (VO₂max: 56±2 ml/kg/min) performed exhaustive cycling exercise on 2 separate occasions. The two trials differed only by the meals ingested after exercise: 1) a high-fat diet designed to maintain energy balance or 2) a low-fat diet designed to elicit energy deficit. The CHO and protein contents of the diets were identical. The next morning we measured plasma substrate and insulin concentrations, CHO oxidation, and muscle biopsies were obtained from the vastus lateralis for measurement of PDK2 and PDK4 mRNA expression, using rt-PCR. Despite identical blood glucose (5.0±0.1 and 4.9±0.1 mM) and insulin (7.9±1.1 and 8.4±0.9 µU/ml) concentrations, plasma fatty acid and glycerol concentrations were elevated 3-4 fold during energy deficit compared with energy balance and CHO oxidation was 40% lower (p<0.01) the morning after energy deficit compared with energy balance (328±69 and 565±89 µmol/min). The lower CHO oxidation was accompanied by a 7.3±2.5 fold increase in PDK4 mRNA expression after energy deficit (p<0.05), while PDK2 mRNA was similar between the trials. In conclusion, energy deficit increases fatty acid availability, increases PDK4 mRNA expression, and suppresses CHO oxidation even when dietary CHO content is not reduced.