Significance Our experiments provide the analysis of lipid metabolite circadian oscillations in a cellular system synchronized in vitro, suggesting cell-autonomous diurnal changes in lipid profiles independent of feeding. Moreover, our work represents a comprehensive comparison between the lipid composition of human skeletal muscle derived from sedentary healthy adults, receiving hourly isocaloric solutions, and human primary skeletal myotubes cultured in vitro. A substantial number of lipid metabolites, in particular membrane lipids, exhibited oscillatory patterns in muscle tissue and in myotube cells, where they were blunted upon cell-autonomous clock disruption. As lipid oscillations in skeletal muscle membrane lipids may impact on insulin signaling and on the development of insulin resistance, studying the temporal lipid composition of human muscle is therefore of utmost importance.