Circadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. We show that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm. Comparison of the diurnal transcriptome and lipidome of human skeletal muscle conducted on serial muscle biopsies in vivo with profiles of human skeletal myotubes synchronized in vitro revealed that ~8% of genes and ~20% of all detected lipid metabolites follow a rhythmic expression both in vivo and in vitro. siCLOCK-mediated clock disruption in primary human myotubes significantly affects the expression of genes related to the glucose homeostasis and lipid metabolism. Moreover, the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes exhibits a circadian profile which was impaired upon siCLOCK. Our findings suggest an essential role for cell-autonomous circadian clocks in coordinating muscle glucose homeostasis, lipid metabolism and myokines secretion in humans.