The characteristics of red blood cells (RBCs) strongly correlate with disease, making measurement techniques valuable for diagnostics. This study focused on RBC deformability and developed an image-based evaluation method using a microfluidic device that applies stress by forcibly stopping flow. Unlike biochemical analyses, such as polymerase chain reaction, which provide averaged data, this method enables single-cell mechanical property assessment. It is also simple, requiring minimal specialized equipment and manual operation. RBCs were confirmed to deform upon forced stopping. We discussed deformation behavior and load estimation, confirming that wall shear stress, not inertial force, dominates deformation. Normal RBCs deformed by 31.5%, whereas adenosine triphosphate (ATP)-depleted RBCs, modeling diseased states, deformed by only 11.1%. The difference was statistically significant (p=0.025). We validated reproducibility by quantifying variability from manual syringe operation, showing consistent deformation measurements across operators. Sensitivity analyses confirmed that assumptions in shear stress estimation, such as gap size and viscosity, had minimal impact. The deformation metric was defined and shown to be robust against image processing variations. Statistical rigor was improved by adding effect sizes, confidence intervals, and power analysis. We discussed the biological relevance and limitations of the ATP depletion model, noting the need for validation with pathological samples. Finally, we highlighted the method’s contribution to intracellular sensor research, positioning mechanical deformability assessment as complementary to molecular intracellular sensing. These results suggest the potential of the method for evaluating blood diseases and related conditions.

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