To study the effect of chronic ketosis on exercise performance in endurance-trained humans, five well-trained cyclists were fed a eucaloric balanced diet (EBD) for one week providing 35-50 kcal/kg/d, 1.75 g protein/kg/d and the remainder of kilocalories as two-thirds carbohydrate (CHO) and one-third fat. This was followed by four weeks of a eucaloric ketogenic diet (EKD), isocaloric and isonitrogenous with the EBD but providing less than 20 g CHO daily. Both diets were appropriately supplemented to meet the recommended daily allowances for vitamins and minerals. Pedal ergometer testing of maximal oxygen uptake (VO2max) was unchanged between the control week (EBD-1) and week 3 of the ketogenic diet (EKD-3). The mean ergometer endurance time for continuous exercise to exhaustion (ENDUR) at 62%-64% of VO2max was 147 minutes at EBD-1 and 151 minutes at EKD-4. The ENDUR steady-state RQ dropped from 0.83 to 0.72 (P less than 0.01) from EBD-1 to EKD-4. In agreement with this were a three-fold drop in glucose oxidation (from 15.1 to 5.1 mg/kg/min, P less than 0.05) and a four-fold reduction in muscle glycogen use (0.61 to 0.13 mmol/kg/min, P less than 0.01). Neither clinical nor biochemical evidence of hypoglycemia was observed during ENDUR at EKD-4. These results indicate that aerobic endurance exercise by well-trained cyclists was not compromised by four weeks of ketosis. This was accomplished by a dramatic physiologic adaptation that conserved limited carbohydrate stores (both glucose and muscle glycogen) and made fat the predominant muscle substrate at this submaximal power level.