High-resolution near-wall fluorescent microparticle image velocimetry (μ-PIV) was used in mouse cremaster muscle venules in vivo to measure velocity profiles in the red cell-depleted plasma layer near the endothelial lining. μ-PIV data of the instantaneous translational speeds and radial positions of fluorescently labeled microspheres (0.47μm) in an optical section through the midsagittal plane of each vessel were used to determine fluid particle translational speeds. Regression of a linear velocity distribution based on near-wall fluid-particle speeds consistently revealed a negative intercept when extrapolated to the vessel wall. Based on a detailed three-dimensional analysis of the local fluid dynamics, we estimate a mean effective thickness of ∼0.33μm for an impermeable endothelial surface layer or ∼0.44μm assuming the lowest hydraulic resistivity of the layer that is consistent with the observed particle motions. The extent of plasma flow retardation through the layer required to be consistent with our μ-PIV data results in near complete attenuation of fluid shear stress on the endothelial-cell surface. These findings confirm the presence of a hydrodynamically effective endothelial surface layer, and emphasize the need to revise previous concepts of leukocyte adhesion, stress transmission to vascular endothelium, permeability, and mechanotransduction mechanisms.