The endothelial glycocalyx is a complex layer of proteoglycans and glycoproteins covering the endothelial surface, playing a crucial role in vascular physiology and pathology. It is involved in mechanotransduction, hemostasis, signaling, and blood cell-vessel wall interactions. The glycocalyx contributes to diabetes, ischemia/reperfusion, and atherosclerosis, and its protective role in the vasculature is supported by experimental data from micro- and macrocirculation. However, reliable visualization of this delicate layer remains challenging. Various techniques, including electron microscopy, two-photon microscopy, and fluorescent labeling, have been used to study the glycocalyx. The glycocalyx is composed of proteoglycans, glycoproteins, and glycosaminoglycans, with heparan sulfate and chondroitin sulfate being the most abundant. The glycocalyx is dynamic, with continuous synthesis and shedding, and its composition and thickness vary with vascular diameter. The glycocalyx influences vascular permeability, blood cell interactions, and endothelial function. It also plays a role in mechanotransduction, sensing shear stress, and modulating inflammatory responses. In pathophysiology, glycocalyx disruption is associated with vascular diseases such as diabetes, ischemia/reperfusion, and atherosclerosis. The glycocalyx is essential for maintaining vascular homeostasis, and its restoration may be a therapeutic target. Visualization techniques, including intravital microscopy and two-photon microscopy, have provided insights into the glycocalyx's structure and function. The glycocalyx is a critical component of the endothelial surface layer, contributing to vascular health and disease.The endothelial glycocalyx is a complex layer of proteoglycans and glycoproteins covering the endothelial surface, playing a crucial role in vascular physiology and pathology. It is involved in mechanotransduction, hemostasis, signaling, and blood cell-vessel wall interactions. The glycocalyx contributes to diabetes, ischemia/reperfusion, and atherosclerosis, and its protective role in the vasculature is supported by experimental data from micro- and macrocirculation. However, reliable visualization of this delicate layer remains challenging. Various techniques, including electron microscopy, two-photon microscopy, and fluorescent labeling, have been used to study the glycocalyx. The glycocalyx is composed of proteoglycans, glycoproteins, and glycosaminoglycans, with heparan sulfate and chondroitin sulfate being the most abundant. The glycocalyx is dynamic, with continuous synthesis and shedding, and its composition and thickness vary with vascular diameter. The glycocalyx influences vascular permeability, blood cell interactions, and endothelial function. It also plays a role in mechanotransduction, sensing shear stress, and modulating inflammatory responses. In pathophysiology, glycocalyx disruption is associated with vascular diseases such as diabetes, ischemia/reperfusion, and atherosclerosis. The glycocalyx is essential for maintaining vascular homeostasis, and its restoration may be a therapeutic target. Visualization techniques, including intravital microscopy and two-photon microscopy, have provided insights into the glycocalyx's structure and function. The glycocalyx is a critical component of the endothelial surface layer, contributing to vascular health and disease.