Simple SummaryDeformation of red blood cells (RBCs) is essential in order to pass through the smallest blood vessels. This cell function is impaired in the presence of high levels of free radicals and shear stress that highly exceeds the physiological range. In contrast, shear stress within the physiological range positively affects RBC function. RBCs are a heterogeneous cell population in terms of RBC age with different RBC deformability described for young and old RBCs, but whether these different sub-populations tolerate mechanical and oxidative stress to the same extent remains unknown. The results of the present investigation revealed lower RBC deformability of old RBCs compared to young RBCs and comparable reductions in RBC deformability of the sub-populations caused by free radicals. Physiological shear stress did not further affect free radical content within the RBCs and reversed the deleterious effects of free radicals on RBC deformability of old RBCs only by improving RBC deformability. The changes were aimed to be explained by changes in the formation of nitric oxide (NO), but outputs of NO generation appeared dependent on cell age. These novel findings highlight a yet less-described complex relation between shear stress, free radicals, and RBC mechanics.Red blood cell (RBC) deformability is an essential component of microcirculatory function that appears to be enhanced by physiological shear stress, while being negatively affected by supraphysiological shears and/or free radical exposure. Given that blood contains RBCs with non-uniform physical properties, whether all cells equivalently tolerate mechanical and oxidative stresses remains poorly understood. We thus partitioned blood into old and young RBCs which were exposed to phenazine methosulfate (PMS) that generates intracellular superoxide and/or specific mechanical stress. Measured RBC deformability was lower in old compared to young RBCs. PMS increased total free radicals in both sub-populations, and RBC deformability decreased accordingly. Shear exposure did not affect reactive species in the sub-populations but reduced RBC nitric oxide synthase (NOS) activation and intriguingly increased RBC deformability in old RBCs. The co-application of PMS and shear exposure also improved cellular deformability in older cells previously exposed to reactive oxygen species (ROS), but not in younger cells. Outputs of NO generation appeared dependent on cell age; in general, stressors applied to younger RBCs tended to induce S-nitrosylation of RBC cytoskeletal proteins, while older RBCs tended to reflect markers of nitrosative stress. We thus present novel findings pertaining to the interplay of mechanical stress and redox metabolism in circulating RBC sub-populations.