The endothelium forms an important part of the vasculature and is involved in promoting an atheroprotective environment via the complementary actions of endothelial cell-derived vasoactive factors. Disruption of vascular homeostasis can lead to the development of endothelial dysfunction which in turn contributes to the early and late stages of atherosclerosis. In recent years an increasing number of non-invasive vascular tests have been developed to assess vascular structure and function in different clinical populations. The present review aims to provide an insight into the anatomy of the vasculature as well as the underlying endothelial cell physiology. In addition, an in-depth overview of the current methods used to assess vascular function and structure is provided as well as their link to certain clinical populations.