PURPOSE: Some recent literature data indicate that ketone bodies inhibit glycolysis in contracting muscles. Therefore, we investigated whether exogenous ketosis by oral ketone ester (KE) intake during prolonged cycling can induce a glycogen sparing action and thereby improve performance during the final phase of the event. METHODS: In a randomized cross-over design, 12 highly-trained male cyclists and triathletes completed a simulated cycling race consisting of 3h submaximal exercise (EX180’) combined with 60g carbohydrates per h, immediately followed by a 15-min time-trial (TT15’) and a maximal sprint test (time to exhaustion at 175% of the anaerobic threshold). Subjects received 20-25g doses of either KE or a non-caloric placebo (CON) at 1h and 20 min before, and at 30 min during EX180’. Blood samples were collected at regular intervals. Biopsies from m. vastus lateralis were obtained before and after EX180’, and at the end of TT15’. RESULTS: KE intake transiently increased blood D-ß-hydroxybutyrate to ~3 mM (range: 2.6-5.2 mM) during EX180’ (p<0.001 vs. CON). Blood pH concomitantly decreased from ~7.42 to 7.36 (range: 7.29-7.40), whilst bicarbonate dropped from 26.0 to 21.6 mM (range: 20.1-23.7) (p<0.001 vs. CON). EX180’ depleted muscle glycogen to the same degree in both groups (KE: -78 ± 9; CON: -60 ± 6 mmol/kg ww, p>0.05). Mean power output during TT15’ (KE: 273 ± 11; CON: 272 ± 11 W, p>0.05) and time-to-exhaustion in the sprint (KE: 59 ± 5; CON: 58 ± 5 s, p>0.05) also were similar between the groups. CONCLUSION: KE intake during a simulated cycling race does not cause glycogen sparing, neither does it affect performance in the final stage of the race.