The endothelial glycocalyx (eGC) is the carbohydrate-rich luminal lining of endothelial cells that mediates permeability and blood cell - vessel wall interactions. The eGC is increasingly considered as an intravascular compartment that protects the vessel wall against pathogenic insults in cardiovascular disease. We hypothesized that the nanomechanical properties of the intact eGC can be measured ex vivo by atomic force microscopy (AFM). Using a mechanical nanosensor, mounted on an atomic force microscope, height (~300 nm) and stiffness (~0.3 pN/nm) of the eGC on the luminal endothelial surface of freshly isolated split-open rat aortas were quantified. Enzymatic removal of heparan sulfate residues by heparinase in vitro led to a dose- and time-dependent reduction of eGC height (–30%) and stiffness (–20%). Similar heparinase-induced changes were found on confluent monolayers of endothelial cell lines and in primary culture. Heparin, a natural substrate for heparinase I, completely abolished these changes. A brief injection of heparinase in healthy rats showed a significant reduction of aortic eGC height (–30%) and stiffness (–30%) compared to controls. Importantly, these changes correlated with increased plasma concentrations of circulating eGC constituents and structural damage of the eGC as evidenced by electron microscopy. Furthermore, AFM reliably detected reduced eGC height and stiffness when applied in an established rat model of chronic kidney disease. In summary, we show that AFM is a valuable tool to quantify endothelial glycocalyx on living endothelial cells ex vivo and in vitro and opens up a mechanical dimension for the investigation of eGC damage in cardiovascular disease.