We determined the effects of a cycle training program in which selected sessions were performed with low muscle glycogen content on training capacity and subsequent endurance performance, whole-body substrate oxidation during submaximal exercise, and several mitochondrial enzymes and signaling proteins with putative roles in promoting training adaptation. Seven endurance-trained cyclists/triathletes trained daily (HIGH) alternating between 100 min steady-state aerobic rides (AT) one day, followed by a high-intensity interval training session (HIT; 8 x 5 min at maximum self-selected effort) the next day. Another 7 subjects trained twice every second day (LOW), first undertaking AT, then 1-2 hr later, the HIT. These training schedules were maintained for 3 wk. Forty-eight hr before and after the first and last training sessions, all subjects completed a 60-min steady-state ride (60SS) followed by a 60-min performance trial. Muscle biopsies were taken before and after 60SS and rates of substrate oxidation were determined throughout this ride. Resting muscle glycogen concentration (412 ± 51 vs. 577±34 µmol•g^-1 d.w.), rates of whole-body fat oxidation during 60SS (1261 ± 247 vs. 1698 ± 174 µmol•kg^-1•60 min^-1), the maximal activities of citrate synthase (45 ± 2 vs. 54 ± 1 mmol•kg^-1•min^-1 d.w) and -hydroxyacyl-CoA-dehydrogenase (18 ± 2 vs. 23 ± 2 mmol•kg^-1•min^-1 d.w) along with the total protein content of cytochrome c oxidase subunit IV were increased only in LOW (all p<0.05). Mitochondrial DNA content and PGC-1 protein levels were unchanged in both groups after training. Cycling performance improved by ~10% in both LOW and HIGH. We conclude that compared to training daily, training twice every second day compromised high-intensity training capacity. While selected markers of training adaptation were enhanced with twice-a-day training, the performance of a 1 hr time-trial undertaken after a 60 min steady-state ride was similar after once daily or twice every second day training programs.