Red blood cell (RBC) aggregation as well as their deformation significantly affects blood microrheology. These processes depend on various factors, one of which is concentration of the nitric oxide, one of the main signaling molecule in the bloodstream. The purpose of this study was to investigate the effect of nitric oxide on the microrheological properties of red blood cells (RBCs) in RBC samples of various media after the addition of nitric oxide donor sodium nitroprusside in vitro. Microrheological properties were measured using laser aggregometer and ektacytometer based on diffuse light scattering and diffraction of laser light on a suspension of RBCs, respectively. The study found that heparin-stabilized blood showed increased RBC aggregation and deformation with sodium nitroprusside concentrations of 100, and 200$$$\mu$M, while EDTA-stabilized blood showed slightly decreased aggregation and unchanged deformation. With washed RBCs in dextran solution, the addition of sodium nitroprusside (in the concentrations of 100, and 200$$$\mu$M) resulted in decreased aggregation and increased deformation. These findings aid in our understanding of nitric oxide’s effect on RBC microrheological properties.