Responses to exchange transfusion using red blood cells (RBCs) with normal and reduced flexibility were studied in the hamster window chamber model during acute moderate isovolemic hemodilution to determine the role of RBC membrane stiffness in microvascular perfusion and tissue oxygenation. Erythrocyte stiffness was increased by 30 min incubation in 0.02% glutaraldehyde solution, and unreacted glutaraldehyde was completely removed. Filtration pressure through 5 µm pore size filters was used to quantify stiffness of the RBCs. Anemic conditions were induced by two isovolemic hemodilution steps using 6% dextran 70 kDa to hematocrit (Hct) of 18% (moderate hemodilution). The protocol continued with an exchange transfusion to reduce native RBCs to 75% of baseline (11% Hct) using either fresh RBCs (RBC) or reduced flexibility RBCs (GRBC) suspended in 5% albumin at 18% Hct; a plasma expander (6% dextran 70k Da, Dex70) was used as control. Systemic parameters, microvascular perfusion, capillary perfusion (functional capillary density, FCD) and oxygen levels across the microvascular network were measured by noninvasive methods. RBC deformability for GRBC was significantly decreased compared to RBC and moderate hemodilution condition. GRBC had a greater mean arterial pressure (MAP) than RBC and Dex70. FCD was substantially higher for RBC (0.81 ± 0.07 of baseline) compared to GRBC (0.32 ± 0.10 of baseline) and Dex70 (0.38 ± 0.10 of baseline). Microvascular tissue pO₂ was significantly lower for Dex70 and GRBC compared to RBC and moderate hemodilution condition. Results were attributed to decreased oxygen uploading in the lungs and obstruction of tissue capillaries by rigidified RBCs, indicating that the effects impairing RBC flexibility are magnified at the microvascular level, where perfusion and oxygenation may define transfusion outcome.