Cryopreservation is a long-term storage strategy for maintaining the quality of red blood cells (RBCs) used for clinical and industrial purposes. However, cryopreservation can induce osmotic stress, leading to a 15 to 20% loss of RBCs during freezing/thawing and cryoprotectant addition/removal. This study investigated how the biological aging of RBCs influences their osmotic characteristics. We hypothesized that biologically older RBCs (O-RBCs) would exhibit diminished osmotic features compared to biologically younger RBCs (Y-RBCs), thereby contributing to their loss during cryopreservation. Seven red cell concentrates (RCCs) were pooled, and their density profile was determined using Percoll separation. Y-RBCs and O-RBCs, representing the extremes of the density spectrum, were isolated. Rheological parameters (O