<p>Blood monitoring in extracorporeal membrane oxygenation (ECMO) is critical to managing the precarious balance between bleeding and thrombosis in ECMO support. Conventional monitoring including plasma free hemoglobin and activated clotting time are typically limited to once-daily measurements. Considering this limitation, we investigated surface zeta (ζ) potential, as a potential real-time biomarker for monitoring red blood cells (RBCs) in ECMO. RBC ζ-potential is a negative electrical charge on the cell surface, providing repulsive force to prevent RBC aggregation in circulation. Isolated human RBCs from 7, 14, 21, and 49 days stored blood were circulated through an ECMO circuit for 12&#xa0;hours. Every 2&#xa0;h, ζ-potential, RBC concentration, intracellular Ca²⁺, and sialic acid were measured. The ζ-potential of RBCs decreased with blood circulation after only 8&#xa0;h of circulation. The decrease in ζ-potential was further amplified by blood storage from just 21 days. Ca²⁺ and sialic acid were also shown to increase with flow and storage duration. This suggests prolonged blood storage compounds the risk of RBC aggregation. Our results demonstrate ζ-potential can detect changes in RBCs under ECMO flow conditions and clinically relevant storage times. The high temporal sensitivity of ζ-potential offers real-time detection of changes in RBC physiology to better predict cell aggregation and clotting risk, improving treatment outcomes for patients.</p>