Introduction: It remains unclear whether exposure to repeated positive acceleration (+ Gz) can exacerbate endothelial dysfunction on the basis of hyperlipidemia. The aim of this study was to investigate the effect of repeated + Gz exposure on endothelial function in high-fat-diet-induced hyperlipidemic rats. Material and methods: Male Sprague-Dawley rats were randomly divided into control, repeat + Gz exposure, high-fat diet (HFD), and + Gz + HFD groups. The rats in the + Gz group were exposed to + Gz and the rats in the HFD group were fed a diet with 2% cholesterol. The rats in the + Gz + HFD group received both the + Gz exposure and HFD. Eight weeks later, the endothelium- dependent relaxation of the aorta was tested and the ultrastructure of the endothelial cells was observed using transmission electron microscopy. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression of endothelial function-associated proteins. Results: Repeated + Gz exposure elevated the serum level of LDL-C in HFD rats. In the + Gz + HFD rats, the ACh-induced relaxation in the aorta rings was significantly attenuated and the endothelial cells of the aorta were dramatically damaged compared with HFD rats. Nitric oxide content and eNOS expression in the aortic tissue were markedly decreased and the oxidative stress was more serious in the + Gz + HFD rats compared with HFD rats. In addition, repeated + Gz exposure significantly increased serum ox-LDL level and LOX-1 expression in the aorta of HFD rats, thereby activating NF-.B p65 and upregulating the expression of interleukin 6, ICAM-1 and VAP-1. Conclusions: Repeated + Gz exposure promotes endothelial dysfunction in HFD-induced hyperlipidemic rats.