Introduction: High performance endurance exercise demands extreme physical loads with carbohydrate intake long accepted as the gold standard for fuelling exercise performance[1]. However, recently we have shown that nutritional ketosis decreases glycolysis[2], increases fat oxidation[3], and remains an oxidisable fuel substrate even at high exercise intensities[4]. Thus we sought to determine whether nutritional ketosis could match improvements elicited by carbohydrate in endurance cycling performance. Methods: Following informed consent, 12 endurance-trained volunteers (n=8 males, VO2 Max 5.4 L/min; n=4 females, VO2 Max 3.8 L/min; age 28.1 y) completed 3 maximal 1h time trials on a bicycle ergometer following an overnight fast, in a 3-way single-blind crossover design. Before each trial isocaloric quantities of either carbohydrate (dextrose; CHO) or ketone ester (725 mg/kg; (R)-3-hydroxybutyl (R)-3-hydroxybutyrate; KE), or a calorie-free control drink and vitamin B3 (1000 mg; B3) were given. Serial blood samples were obtained via an IV catheter and analysed for glucose, free fatty acids (FFA), D-β-hydroxybutyrate, lactate, glycerol, insulin and acetoacetate. Muscle biopsies (vastus lateralis) were obtained from n=7 athletes pre- and immediately post- exercise. Samples were cryo-sectioned, stained, and analysed for glycogen+ intramuscular triglyceride (IMTAG) using confocal microscopy. 3-way repeated measures ANOVA with post-Hoc Tukey corrections were used to determine statistical significance (p<0.05). Results: Power output during the time trial was improved with both CHO and KE supplementation compared to vitamin B3 (B3 284±6 W; CHO 295±5 W p=0.01; KE 292±6 W p=0.01), with no difference observed between CHO and KE. Blood D-β-hydroxybutyrate reached a mean of 3.2± 0.2 mM prior to the time trial following KE ingestion remaining elevated throughout exercise. There was a significant increase in blood glucose and insulin in CHO vs. KE and B3, while lactate production was lower during KE vs. both CHO and B3 (p<0.05). Plasma FFA were similar during exercise on B3 and KE, while a significant increase was observed in the latter half of exercise with CHO supplementation (p<0.05). IMTAG and glycogen were not significantly different between conditions at baseline, however IMTAG fell by 8% on KE but increased 9% on CHO (p<0.05) after 1 h of exercise. IMTAG was unchanged on B3. Glycogen levels were significantly greater after exercise on KE vs. CHO and B3 (p<0.05) indicative of glycogen preservation by ketosis. Conclusion: In this study we have shown how nutritional ketosis enables equivalent physiological function and cycling performance to that of glucose, but via very different metabolic actions. Preservation of physiological function is very much in keeping with survival metabolism, where maintenance of homeostasis during conditions of altered fuel availability is vital[5].