It is well known that the morphology of endothelial cells in culture changes dramatically as they go from subconfluence to confluence. After reaching confluence, however, the morphology continues to change but much more subtly as cell density increases and they become more uniform in size and shape. Measurements of surface topography of confluent cells by atomic force microscopy (AFM) showed that cell heights became more uniform and that the root-mean-square amplitude of surface undulations decreased by 7% compared with monolayers that had just reached confluence. Computational fluid dynamics simulations of flow over the endothelial surface geometries measured by AFM showed that the change in topography with time after confluence altered the shear stress distribution, resulting in an increase in the stress concentrations experienced by the cells despite the reduced amplitude of the surface undulation. These data suggest that the starting point for in vitro experiments may influence the measured responses to shear stress, particularly transient responses that occur before structural adaptation takes place. In addition, changes in surface topography may reflect changes in cell tension, cytoskeletal structure, and adhesion to the substratum, all of which are associated with the regulation of growth in anchorage-dependent cells.