Recruitment of fatty acids from adipose tissue is essential during fasting. However, the molecular mechanisms behind fasting-induced metabolic regulation in human adipose tissue and the potential impact of training state in this are unknown. Therefore, the aim of the present study was to investigate 1) fasting-induced regulation of lipolysis and glyceroneogenesis in human adipose tissue as well as 2) the impact of training state on basal oxidative capacity and fasting-induced metabolic regulation in human adipose tissue. Untrained (VO2max < 45ml·min-1·kg-1) and trained subjects (VO2max > 55ml·min-1·kg-1) fasted for 36h and abdominal subcutaneous adipose tissue biopsies were obtained 2, 12, 24 and 36h after a standardized meal. Adipose tissue OXPHOS, phosphoenolpyruvate carboxykinase and pyruvate dehydrogenase (PDH) E1α protein as well as PDH kinase (PDK) 2, PDK4 and PDH phosphatase 2 mRNA content were higher in trained subjects than untrained subjects. In addition, trained subjects had higher adipose tissue hormone sensitive lipase Ser660 phosphorylation and adipose triglyceride lipase protein content as well as higher plasma free fatty acids concentration than untrained subjects during fasting. Moreover, adipose tissue PDH phosphorylation increased with fasting only in trained subjects. Taken together, trained subjects seem to possess higher basal adipose tissue oxidative capacity as well as higher capacity for regulation of lipolysis, glyceroneogenesis and substrate availability in adipose tissue than untrained subjects.