Abstract: To investigate the coupled effects of storage duration and cyclic tensile loading on the viscoelasticity and deformability of red blood cells (RBCs), a dielectrophoresis-based microfluidic platform for single-cell mechanical characterization was developed. RBCs stored for different durations (0, 4, 7, and 13 days) were subjected to controlled cyclic electrical deformation (period of 4 s, effective voltage of 2 V). Using the Kelvin-Voigt viscoelastic model, the deformation ratio–time curves were piecewise fitted with exponential functions to quantitatively determine the changes in shear modulus and shear viscosity of the RBC membrane. The results show that as the number of cyclic stretching cycles increases, the maximum deformation ratio of RBCs continuously decreases. Both shear modulus and shear viscosity increase monotonically, with the shear modulus in the 13-day storage group rising to 5–6 times its initial value after 2,250 cycles. Storage duration significantly affects the degradation rate of RBC mechanical properties: short-term storage (0–7 days) results in relatively mild changes in mechanical parameters, while long-term storage (13 days) markedly enhances fatigue sensitivity, with the shear modulus increasing by more than 250%. This study reveals that cumulative membrane fatigue hardening induced by cyclic stretching is the primary cause of the decline in RBC deformability, and that storage duration accelerates this process by reducing membrane structural stability, offering a new perspective for understanding the mechanical adaptation mechanism of RBCs in blood circulation.