Ceramide and diacylglycerol are linked to insulin resistance in rodents, but in humans the data are inconsistent. Insulin resistance is frequently observed with aging, but the role of ceramide and diacylglycerol is not clarified. Training improves metabolic health and, therefore, we aimed to elucidate the influence of age and high-intensity interval training (HIIT) on ceramide and diacylglycerol content in muscle. Fourteen young (33 ± 1) and 22 older (63 ± 1) overweight to obese subjects performed 6 weeks HIIT three times a week. Maximal oxygen uptake and body composition were measured and muscle biopsies and fasting blood samples were obtained. Muscle ceramide and diacylglycerol were measured by gas-liquid chromatography and proteins in insulin signaling, lipid and glucose metabolism were measured by Western blotting. Content of ceramide and diacylglycerol total, saturated, C16:0 and C18:0 fatty acids and C18:1 ceramide were higher in older compared to young. HIIT reduced saturated and C18:0 ceramides, while the content of the proteins involved in glucose (GLUT4, glycogen synthase, hexokinase II, AKT) and lipid metabolism (adipose triglyceride lipase, fatty acid binding protein) were increased after HIIT. We demonstrate a higher content of saturated ceramide and diacylglycerol fatty acids in the muscle of older subjects compared to young. Moreover, the content of saturated ceramides was reduced and muscle glucose metabolism improved at protein level after HIIT. This study highlights an increased content of saturated ceramides in aging which could be speculated to influence insulin sensitivity.