The healthy endothelium secretes and expresses at the surface various molecules which help maintain vascular wall structure and vascular homeostasis, as well as regulating vascular tone and leukocyte traffic. In response to various aggressive stimuli, the endothelial cell modulates its properties to restore vascular homeostasis. Usually, changes in the endothelial cell phenotype are transient and do not compromise the subsequent re-establishment of endothelial function. However, in certain pathological conditions, such as atherosclerosis, endothelial function is chronically disturbed, and this alteration is a critical step in the progression of the disease. In recent years, advances in knowledge have improved our understanding of the physiopathology of atherosclerosis, which is now known to be a dynamic and progressive process proceeding from endothelial dysfunction and inflammation of the vascular wall. The evolution and prognosis of atherosclerosis, along with the efficacy of therapeutic modalities, can be assessed by measuring the circulating levels of various biomarkers expressed or released by the endothelium. The purpose of this review is to reappraise the evidene concerning endothelial function under physiological conditions and in atherosclerosis, and to analyze markers of endothelial function with clinical applicability.