Calorie restriction (CR) is accepted as an experimental anti-aging paradigm. Several important signal transduction pathways including AMPK and SIRT1 are implicated in the regulation of physiological processes of CR. However, the mechanisms responsible for adaptations remain unclear in humans.Four overweight male participants were enrolled and treated with 25% CR of their baseline energy requirements for 7weeks. Characteristics, including body weight (BW), body mass index (BMI), %fat, visceral fat area (VFA), mean blood pressure (MBP) and VO2 max, as well as metabolic parameters, such as insulin, lipid profiles and inflammatory makers and the expression of phosphorylated AMPK and SIRT1 in peripheral blood mononuclear cells (PBMNCs), were determined at baseline and then after 7weeks. In addition, we assessed the effects of the serum collected from the participants on AMPK and SIRT1 activation and mitochondrial biogenesis in cultured human skeletal muscle cells.After CR, BW, BMI, %fat, VFA and MBP all significantly decreased, while VO2 max increased, compared to those at baseline. The levels of fasting insulin, free fatty acid, and inflammatory makers, such as interleukin-6 and visfatin, were significantly reduced, whereas the expression of phosphorylated AMPK and SIRT1 was significantly increased in PBMNCs collected after CR, compared to those at baseline. The skeletal muscle cells that were cultured in serum collected after CR showed an increase in AMPK and SIRT1 activity as well as mitochondrial biogenesis.CR is beneficial for obesity-related metabolic alterations and induces cellular adaptations against aging, possibly through AMPK and SIRT1 activation via circulating factors.