Ischemia reperfusion (IR) lesions are an unavoidable consequence of organ transplantation. Researching new therapeutics against these lesions requires the definition of early mechanisms. The cytoskeleton is composed of 3 types of filaments: microfilaments, intermediate filaments, and microtubules. We aimed to characterize the influence of preservation on their phenotype. In an in vitro model using primary human endothelial cells reproducing the conditions of organ preservation, two aspects were explored: (a) the impact of IR and cold ischemia time on each filament type, evaluating the roles of temperature, solution, and oxygen; and (b) the potential of cytoskeleton-mediated therapy to alleviate cell death. Results showed that intermediary filaments were unaffected, while microfilaments showed radical changes with disappearance of the structure replaced by a disorganized array of nodules; moreover, microtubules almost completely disappeared with time. Furthermore, temperature, and not oxygen deprivation or the solution, was the determining factor of the cytoskeleton's loss of integrity during preservation. Finally, pharmaceutical intervention could indeed preserve fiber structure but did not alter survival. Our work shows that improvement of preservation must include a more adapted temperature before considering oxygen, as it could profoundly improve cytoskeleton organization and thus cell fate. This highlights the importance of this structure for the development of new therapeutics and the definition of graft quality biomarkers.