Introduction Ketone bodies act as both substrates and signals to conserve carbohydrates for cerebral oxidation during calorie deprivation[1]. Administration of exogenous ketones alters skeletal muscle substrate competition for respiration in exercise[2, 3], however the magnitude of this effect in relation to skeletal muscle fibre type composition is unknown. Methods After providing informed consent, and after an overnight fast, n=7 trained male volunteers (age 29.7 y, VO2max 4.8 L/min) drank 1.14 g/kg BW of ketone ester ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate; KE) and dextrose, or isocaloric carbohydrates (CHO), in a randomised, blinded, crossover design. After 30 min, athletes performed 2 h of bicycle exercise at a fixed intensity of 70% VO2 Max. Blood samples were obtained via an intravenous catheter at regular intervals during exercise, and assayed for β-hydroxybutyrate (BHB), lactate, glucose, and free fatty acids (FFA). Indirect calorimetry (Cortex, Metalyser) was concurrently performed to quantify respiratory quotient (RQ) and VO2. Muscle biopsies of the vastus lateralis were obtained before and after exercise. Muscle samples were cryosectioned, stained for intramuscular triacylglycerol (IMTAG), glycogen, and fibre type, and quantified using confocal microscopy. A 2-way repeated measures ANOVA with post-Hoc Tukey correction, or 2-tailed Pearson correlations were used to determine statistical significance (considered as p<0.05). Results Blood BHB rose from 0.1 to 2.2 mM (p<0.01) following KE ingestion, reaching 3.2 mM after 2 h of exercise. FFA were identical for the first 60 min of exercise but increased on CHO vs. KE by 2 h (p<0.05). Mean exercise RQ was significantly lower on KE vs. CHO (0.89 ± 0.02 vs. 0.97 ± 0.02, p<0.05). IMTAG and glycogen were not significantly different between conditions at baseline, however IMTAG fell by 24% on KE vs. 1% on CHO (p<0.01) after 2 h of exercise. There was a direct relationship between IMTAG oxidation and % of slow type muscle fibre content (r = 0.65, p<0.05) and commensurate reduction in glycogenolysis (p<0.05) during ketosis. However the direct opposite relationship was observed on CHO with an increase in type II muscle fibre content associated with higher lipid storage during exercise, and a reduction in muscle glycogen levels. Overall there was a curvilinear reciprocal relationship between glycogen use in exercise and IMTAG oxidation (r =0.8 p<0.01). Conclusion Nutritional ketosis is able to harness the innate metabolic response to starvation, increasing IMTAG oxidation during exercise in the presence of normal muscle glycogen and co-ingested carbohydrate. These data suggest that the metabolic effects of glycogen preservation and increased IMTAG oxidation during ketosis are accentuated in athletes with greater (oxidative) slow twitch muscle fibre content.