This pilot study evaluated how cardiopulmonary bypass (CPB), with and without hypothermic circulatory arrest (HCA), affects red blood cell (RBC) morphology, membrane nanostructure, and mechanical properties using atomic force microscopy. In 14 cardiac surgery patients, CPB increased RBC stiffness, while CPB+HCA caused more severe morphological damage and membrane roughness. The findings suggest distinct cellular injury mechanisms depending on perfusion strategy.