The distribution of hemodynamic shear stress throughout the arterial tree is transduced by the endothelium into local cellular responses that regulate vasoactivity, vessel wall remodeling, and atherogenesis. Although the exact mechanisms of mechanotransduction remain unknown, the endothelial cytoskeleton has been implicated in transmitting extracellular force to cytoplasmic sites of signal generation via connections to the lumenal, intercellular, and basal surfaces. Direct observation of intermediate filament (IF) displacement in cells expressing green fluorescent protein-vimentin has suggested that cytoskeletal mechanics are rapidly altered by the onset of fluid shear stress. Here, restored images from time-lapse optical sectioning fluorescence microscopy were analyzed as a four-dimensional intensity distribution function that represented IF positions. A displacement index, related to the product moment correlation coefficient as a function of time and subcellular spatial location, demonstrated patterns of IF displacement within endothelial cells in a confluent monolayer. Flow onset induced a significant increase in IF displacement above the nucleus compared with that measured near the coverslip surface, and displacement downstream from the nucleus was larger than in upstream areas. Furthermore, coordinated displacement of IF near the edges of adjacent cells suggested the existence of mechanical continuity between cells. Thus, quantitative analysis of the spatiotemporal patterns of flow-induced IF displacement suggests redistribution of intracellular force in response to alterations in hemodynamic shear stress acting at the lumenal surface.