The mechanical condition in stack assembly can impose a significant influence in performance and reliability of the vanadium flow battery. In order to analyze the influence and optimize the overall performance, the effect of clamping force on electrode morphology, mass transfer and polarizations is investigated in this study by measurement of contact resistance and development of a coupled mechanical-electrochemical model. The experimental results indicate that the contact resistance and ohmic polarization decreases with an increase in clamping force, while the simulation results demonstrate that the clamping force can significantly impact on the electrode morphology and mass transport phenomena, and subsequently affect the distributions of activation and concentration polarizations. Despite the concentration polarization presenting to rise at a large clamping force, the overall polarization is proved to be decreased demonstrating that an increased clamping force can offer high voltage efficiency and power density. By further considering the mechanical failure probability and trading off the cell performance and reliability, an optimal clamping force is successfully determined. Such an analysis focusing on assembling condition is vital for understanding the effect of mechanical condition on the battery performance, while also highlighting the significance of rational mechanical design and assembly in flow battery manufacture.