Endothelial cells, master gatekeepers of the cardiovascular system, line its inner boundary from the heart to distant capillaries constantly exposed to blood flow. Inter-endothelial signaling and the monolayer's adhesion to the underlying collagen rich basal lamina are key in physiology and disease. Using neutron scattering, we report the first-ever interfacial structure of endothelial monolayers under dynamic flow conditions mimicking the cardiovascular system. Endothelial adhesion strength (defined as the separation distance l between the basal cell membrane and solid boundary) is explained using developed interfacial potentials and intra-membrane segregation of specific adhesion proteins. Our method provides a powerful tool for the biophysical study of cellular layer adhesion strength in living tissues.