Hypothermic machine perfusion preservation (MPP) has the potential to relieve the current donor shortage problem by providing superior preserved tissue and viable non-heart-beating donor tissue. For the liver, MPP has not improved preservation. Currently, the major cause of damage associated with MPP of livers is unknown. An intravital microscopy study was conducted to investigate the state of sinusoidal perfusion during 24-hour MPP. Fluorescein isothiocynate (FITC)-labeled albumin was utilized to mark the microvascular space while FITC-labeled red blood cells were used to determine the fluid velocity. The results showed that there was an increase in vascular resistance (> 275%) when the liver was perfused with a UW solution for 24 hours at 5°C and with a flow rate of 5 ml/min. This vascular resistance further increased (> 425%) during rewarming (for 1 hour, at 37°C and 15 ml/min). The mean flow velocities increased during initial MPP from 236 ±16 μm/s (mean ± standard error) to 434 ± 20 μm/s and the mean shear stress values increased from 5.3 ± 0.8 dynes/cm2 to 6.5 ± 0.8 dynes/cm2, after 24 hours of MPP the mean flow velocity values and shear stress values decreased (223 ± 13 μm/s and 3.3 ± 0.8 dynes/cm2) respectively. The reason for this was detected by the FITC-labeled albumin, in the tissue. It was evident that these areas (after 24 hours of MPP) also displayed increased blockage. It also appeared from the micrographs and the histology study that the blockage occurred as a result of endothelial cells rounding after 24 hours of MPP. The cells remained rounded even after rewarming the tissue. This could be a mechanism of damage to the liver during 24-hour of MPP.