Vascular endothelial cells (ECs) respond to temporal and spatial characteristics of hemodynamic forces by alterations in their adhesiveness to leukocytes, secretion of vasodilators, and permeability to blood-borne constituents. These physiological and patho-physiological changes are tied to adaptation of cell mechanics and mechanotransduction, the process by which cells convert forces to intracellular biochemical signals. The exact time scales of these mechanical adaptations, however, remain unknown. We used particle tracking microrheology to study adaptive changes in intracellular mechanics in response to a step change in fluid shear stress, which simulates both rapid temporal and steady features of hemodynamic forces. Results indicate that endothelial cells become significantly more compliant as early as 30 seconds after step change in shear stress from 0 to 10 dynes/cm² followed by recovery of viscoelastic parameters within 4 minutes of shearing even though shear stress was maintained. After 5 minutes of shearing, return of shear stress to 0 dynes/cm² in a stepwise manner did not result in any further rheological adaptation. Average vesicle displacements were also used to determine time-dependent cell deformation and macrorheological parameters by fitting creep function to a linear viscoelastic liquid model. Characteristic time and magnitude for shear-induced deformation were 3 seconds and 50 nm, respectively. We conclude that endothelial cells rapidly adapt their mechanical properties in response to shear stress and we provide the first macrorheological parameters for time-dependent deformations of ECs to a physiological forcing function. Such studies provide insight into pathologies such as atherosclerosis which may find their origins in EC mechanics.