Human muscle is studied during glycogen depletion and repletion to understand the influence of exercise and muscle glycogen on total ceramide content. In addition, fiber-type-specific ceramide storage is investigated. Ten healthy males (26.4 ± 0.9 years, BMI 24.4 ± 0.7 kg m−2 and VO2max 57 ± 2 mL O2 min−1 kg−1) participated in the study. On the first day, one leg was glycogen-depleted (DL) by exhaustive intermittent exercise followed by low carbohydrate diet. Next day, in the overnight fasted condition, muscle biopsies were excised from vastus lateralis before and after exhaustive exercise from both DL and control leg (CL). Muscle glycogen was analyzed biochemically and total muscle ceramide content by 2D quantitative lipidomic approach. Furthermore, fiber-type ceramide content was determined by fluorescence immunohistochemistry. Basal muscle glycogen was decreased (P <0.05) with 50 ± 6% in DL versus CL. After exhaustive exercise, muscle glycogen was similar in CL and DL 139 ± 38 and 110 ± 31 mmol kg−1, respectively. Total muscle ceramide 58 ± 1 pmol mg−1 was not influenced by glycogen or exercise. Ceramide content was consistently higher (P <0.001) in type I than in type II muscle fibers. In conclusion, human skeletal muscle, ceramide content is higher in type I than in type II. Despite rather large changes in muscle glycogen induced by prior depletion, exercise to exhaustion and repletion, total muscle ceramide concentration remained unchanged.