Endothelial function is modulated by wall shear stress acting on the vessel wall which is determined by the fluid velocity and the local viscosity near vessel wall. Red blood cell (RBC) aggregation may affect the local viscosity by favoring axial migration. The aim of this study was to investigate the role of RBC aggregation, with or without altered plasma viscosity, in the mechanically-induced nitric oxide (NO) related mechanisms of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured on the inner surface of cylindrical glass capillaries which were perfused using RBC suspensions having normal and increased aggregation at a nominal shear stress of 15 dyn/cm². RBC aggregation was enhanced using two different approaches: 1) Poloxamer coated RBC, suspended in normal, autologous plasma resulting in enhanced aggregation but unchanged plasma viscosity; 2) Normal RBC suspended in autologous plasma containing 0.5% dextran (MW: 500 kD), with similar level of RBC aggregation and but higher plasma viscosity. Compared to normal cells in unmodified plasma, perfusion with suspensions of poloxamer-coated RBC in normal plasma resulted in decreased levels of NO metabolites and serine 1177 phosphorylation of eNOS. Perfusion with normal RBC in plasma containing dextran resulted in NO level that remained elevated, whereas only a modest decrease of phosphorylated eNOS level was observed. The results of this study suggest that increases of RBC aggregation tendency affect endothelial cell functions via altering local blood composition, especially if the alterations of RBC aggregation are due to modified cellular properties and not to plasma composition changes.