The endothelial glycocalyx (eGC) is an anionic biopolymer layer that covers the luminal side of blood vessels. There is accumulating evidence that the eGC is a dynamic structure which is crucial for maintaining normal vascular function. Still, only little is known about the eGC function due to lack of experimental approaches. There we tried to establish a method based on atomic force microscopy (AFM) characterizing the mechanical properties of eGC on living cells in real time. As a 'proof of principle' we simultaneously used fluorescence microscopy (FM) on the same cells by means of a combined AFM/FM setup. Experiments were performed in living GM7373 endothelial cells cultured in vitro. For AFM indentation measurements we used soft cantilevers with spherical tips. For FM we immunostained heparan sulfate proteoglycans with Qdot-labeled antibodies. Analysis of the data revealed a soft layer on the surface of endothelial cells with a height of about 180 nm and a stiffness of about 0.23 pN/nm which is about 4 times softer compared to the cell membrane. Heparinase treatment leads to the reduction of eGC-stiffness by 43%, of eGC-height by 47% and Qdots abundance of the cell surface by 40%. The decrease of surface-bound Qdots correlates strongly with the decrease in eGC-stiffness (Spearman Correlation Coefficient = 0.78) and the decrease in eGC-height (Spearman Correlation Coefficient = 0.81). This indicates that the measured surface layer is most likely the endothelial glycocalyx. In conclusion, a method was established to characterize the nanomechanical properties of the eGC on living cells.