The demand for organs has significantly increased pressure on procurement centers, to the detriment of organ quality. To face the increasing complications rate, new preservation and evaluation methods for marginal organs are urgently needed. However, such advances require in depth understanding of ischemia reperfusion mechanisms. We performed a LC-MS/MS proteomic analysis of cold ischemia’s impact on endothelial cells, key cell type of this injury, during hypothermia (every 3 hours) and after reperfusion. We established that key proteins of critical pathways involved in energy metabolism, cytoskeleton structure and transport system and gene transcription and translation were dynamically altered in response to cold ischemia time (CIT). Key time zones were revealed: a-during the first 3 hours, key proteins were upregulated within these pathways; b-the majority of these upregulation were maintained until 12 hours CIT; c-after that time, overall decrease in protein expression was observed until the end of CIT; d-at reperfusion, proteins expressed in response to cold ischemia were all downregulated. Our results show that cold ischemia is not a simple slowing down of the cell metabolism and inhibition of lesions. Deep changes take place within the proteome on major pathways, including protein producing machinery. In particular, the cell’s response to CIT indicate a capability to resist stresses for a time. In depth investigation of these pathways could uncover new strategies to maintain organ quality, or optimize it, as well as uncover key biomarkers to monitor stresses.