Recent studies indicate that exercise with a low muscle glycogen state enhances exercise-induced metabolic adaptation. However, it is unclear whether metabolic adaptation is involved with muscle glycogen depletion level. In this study, we investigated the effects of prior muscle glycogen depletion level on metabolic response during acute continuous exercise. Seven men completed two experimental trials consisting of two exercise sessions per day. During the first session, participants performed either intermittent exercise (IE) at VO₂max (the IE-CE trial) or continuous exercise (CE) at lactate threshold (the CE-CE trial). During the second session, participants performed 60 minutes of CE at lactate threshold. During this second session, fatty acid oxidation (FAO) was calculated. To determine muscle glycogen content and PGC-1α and PDK-4 mRNA abundance, muscle biopsies were taken at rest after the first session and 2 hours after the second session. After the first session, muscle glycogen content was significantly lower in the IE-CE trial (38.1±5.0 mmol/kg w.w.) than in the CE-CE trial (56.7±10.2 mmol/kg w.w.), P<0.05. FAO was higher in the IE-CE trial than the CE-CE trial at baseline and 15 minutes after the second session (both P<0.05). PGC-1α mRNA abundance increased after exercise (IE-CE, 5.9±2.5; CE-CE, 2.6±1.3-fold; P<0.1). PDK-4 mRNA abundance increased significantly after exercise (IE-CE, 22.2±8.8; CE-CE, 31.5±10.6-fold; P<0.05). PGC-1α and PDK-4 mRNA were not significantly different between the trials. In conclusion, continuous exercise with a slightly muscle glycogen-depleted state induced similar level of PGC-1α and PDK-4 mRNA expression, but attenuated FAO, compared to exercise with a moderate muscle glycogen-depleted state.