Shear stress is one of the major hemodynamic forces acting on the endothelium. However, it is not well known how endothelial cells (EC) respond mechanically to these stimuli in vivo. Here we investigated whether changes in biomechanics properties and shear stress could increase cell susceptibility to injury, contributing to vascular fragility. We surgically implanted a shear stress modifier device on the carotid artery of ApoE-knockout mice (ApoE−/−), which, due to its shape, causes a gradual stenosis in the vessel, resulting in distinct shear stress patterns. Our data show actin fibers accumulation in areas with higher lipid deposition in ApoE−/−, indicating that dyslipidemia might interfere with EC actin cytoskeleton organization. We also showed that both shear stress and dyslipidemia were important for EC susceptibility to injury. Furthermore, lysosomal distribution, an important organelle for plasma membrane repair, was altered in ApoE−/−, which could compromise EC’s ability to repair from damage. Therefore, dyslipidemia and variations in shear stress patterns not only affect cellular mechanics by compromising the actin cytoskeleton organization, but also enhance cell susceptibility to injury and alter vesicle trafficking in vascular cells. This may likely contribute to vascular fragility and thus to the initial steps of atherosclerosis development.