Introduction: We developed a nutritional ketone ester drink that mimics the physiology of starvation by providing ketones as a fuel source[1]. Ketone bodies have energetic advantages over other fuel substrates, and may alter oxidative fuel selection in working muscle due to their evolutionary role in conserving glucose[2]. We tested whether ketone bodies could alter glycolysis during highly glycolytic exercise; a physiological state of metabolic inflexibility where glucose is the preferred substrate[3]. Methods: After providing informed consent, and following an overnight fast, 10 highly trained male volunteers (age 28.4 y, VO2 max 5.4 L/min) drank isocaloric quantities of carbohydrate (CHO), fat (FAT), or ketone ester (KE)(0.573 g/kg BW), in a randomised blinded crossover design. After 25 min, athletes performed 1 hour of bicycle exercise at 75% Wmax. Muscle biopsies of the Vastus Lateralis were obtained before and after exercise. Muscle metabolites were extracted via the Folsch method, and analysed using a triple quadrupolar mass spectrometer (Waters, UK). 3-way repeated measures ANOVA with post-Hoc Tukey corrections were used to determine statistical significance (considered as p<0.05). Results: Blood ketones rose from 0.1 to 3.4 mM (p<0.01) following ketone drinks. Intramuscular ketone concentrations were 3 fold greater on KE vs. FAT/ CHO. Intramuscular glycolytic intermediates, glyceraldehyde-3-phosphate, 2&3-phosphoglycerate and pyruvate, were significantly lower on KE vs. CHO/FAT (p<0.05). Fructose-1,6-bisphosphate, dihydroxyacetone phosphate (DHAP), and 1,3-bisphosphoglycerate were the same at rest. After 60 min exercise at 75% Wmax the sum of intramuscular glycolytic intermediates increased 1-2 fold over the resting conditions (p<0.05). However, with the exception of DHAP, intramuscular glycolytic intermediates were ~40% lower on KE vs. FAT/CHO (p<0.05) at the same workload, whereas FAT vs. CHO were not different. Conclusion: Nutritional ketosis represents a novel physiological state, providing a new fuel for oxidative respiration, whilst decreasing muscle glycolysis during highly glycolytic exercise. This suggests that re-evaluation of the hierarchy of muscular fuel selection in exercise is required in the presence of ketosis. Acute nutritional ketosis may have utility in other physiological or pathological conditions where glycolytic demands are high and substrate energetic performance is paramount.