High body mass index- (BMI-) related vascular injury contributes to the pathogenesis of the atherosclerotic cardiovascular disease (ASCVD). Rigorous calorie restriction is one of the major lifestyle interventions to reduce vascular risk in overweight or obese individuals. However, the effects of fasting therapy (FT) on vascular function and the mechanism are still unclear. This study was aimed to investigate the impacts of FT on endothelial function, arterial stiffness, and circulating arterial damage parameters in overweight and obese individuals and possible mechanism. Overweight and obese individuals participated in FT intervention (7-day very low calorie diet). Arterial function including brachial arterial flow-mediated dilation (FMD), brachial-ankle pulse wave velocity (baPWV), vascular injury-related markers including trimethylamine N-oxide (TMAO), and leptin and endothelial microparticles (EMPs) were assessed. Endothelial progenitor cells (EPCs) of these participants were isolated and cultured to investigate EPCs function. mRFP-GFP-LC3 confocal microscopy scanning and western blot were carried out to determine autophagy. After FT, body weight and BMI significantly decreased (81.76 ± 12.04 vs. 77.51 ± 12.06 kg, P < 0.01; 29.93 ± 2.82 vs. 28.47 ± 2.83 kg/m2, P < 0.01). FT remarkably improved FMD (5.26 ± 1.34 vs. 6.25 ± 1.60%, P=0.01) while baPWV kept unchanged. TMAO and leptin levels decreased (3.96 ± 1.85 vs. 2.73 ± 1.33 μmol/L, P=0.044; 6814 ± 2639 vs. 3563 ± 2668 μmol/L, P < 0.01). EMPs showed a decreased tendency. EPCs function was significantly improved, autophagy fluorescence intensity was enhanced, and the level of Beclin1, Atg5, LC3 II/I also increased after starvation in vitro, and the effects were blocked by autophagy inhibitor. Our present study demonstrated for the first time that FT markedly improves endothelial function and reduces the levels of arterial injury markers through improving EPCs function via activating autophagy. These findings provide a novel insight into FT as a lifestyle intervention strategy to promote the maintenance of vascular homeostasis in overweight or obese individuals. The trial was registered with ChiCTR1900024290. Copyright © 2020 Jiapan Sun et al.