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Background/Aims: In vitro studies have shown that band-3 function is mainly regulated by its phosphorylation status. The main purpose of the study was to investigate whether band-3 phosphorylation status interferes with an exhaustive running exercise-related dysfunction of RBC deformability. Methods: Rats were divided into sedentary control (C) and exercise test (ET) groups. The ET group was divided further into exhaustive running exercise (ERE) and moderate running exercise (MRE) subgroups. Results: Tyrosine phosphorylation of band-3 was significantly elevated in the absence of reducing agent, consistent with the emergence of band-3 clustering in the ERE group compared with the control and MRE groups. The elongation index (EI) was found to decline significantly in the ERE group compared with the C and MRE groups under shear stress (control group, 0.41 ± 0.01 at 3 Pa and 0.571 ± 0.008 at 30 Pa; ERE group, 0.3140 ± 0.013 at 3 Pa and 0.534 ± 0.009 at 30 Pa; P Conclusion: Our results suggest that exhaustive running exercise results in elevated band-3 tyrosine phosphorylation and alters band-3 membrane organization. Furthermore, it appears that exhaustive running exercise induced band 3 phosphorylation is due to the oxidation of critical sulfydryl groups of a membrane phosphatase (PTP).