Accurate characterization of the mechanical properties of brain tissue is essential for understanding the mechanisms of traumatic brain injuries and developing protective gears or facilities. However, how storage conditions might affect the mechanical properties of brain tissue remains unclear. The objective of this study is to investigate the effect of in vitro storage duration on the mechanical performance of brain tissue since measurements are usually carried out in vitro. Differential Scanning Calorimetry (DSC) measurements and uniaxial compression mechanical experiments are carried out. The results indicate that, for brain tissue stored at 1 °C without any liquid medium, the bio-molecular interactions and the mechanical strength of both white and grey matter deteriorate with prolonged storage duration. Transmission Electron Microscopy (TEM) results reveal the degeneration of myelin sheaths and the vacuolization of cristae with prolonged storage duration, suggesting that the in vitro storage duration should be carefully controlled. The findings from this study might facilitate the development of guidelines and standards for the in vitro storage of brain tissue.