Objective To improve the availability of vessel grafts for allotransplantation, the current experimental study was designed to first investigate the function of vessels obtained from non-heartbeating donor rats at various time points postmortem; second, to assess the sensitivity of vessels recovered after circulatory arrest toward prolonged cold storage; and third, to determine vessel function following cold storage with antimicrobial additives. Methods We investigated vessel tone development and endothelium-dependent and endothelium-independent relaxations in a Mulvany myograph of aorta and saphenous artery sampled up to 24 hours after circulatory arrest. Additionally, tissue reductive capacity and lactate dehydrogenase release were measured. Results Vessels recovered 2 hours postmortem showed similar results as controls recovered without delay. Vessels recovered 6 hours or more after circulatory arrest showed reduced vessel tone development (ie, aorta): response to potassium <15% and response to norepinephrine <25% of vessels recovered without delay; A. saphena response to potassium: <12% and response to norepinephrine <10% of control vessels recovered without delay. All vessels recovered after circulatory arrest showed a similar cold storage sensitivity as controls, with exception of a decreased endothelial function of A. saphena harvested 6 hours postmortem (one-third response of non-stored control vessels). Treatment of vessels recovered immediately or after circulatory arrest with gentamycin, piperacillin, and metronidazole as additives to the optimized cold storage solution did not alter vessel function. Flucloxacillin as a cold storage additive reduced vessel tone development in aorta but not in A. saphena. Addition of amphotericin B to the storage solution completely abolished any vessel function and impaired tissue reductive capacity despite presence of radical scavengers. Conclusions The use of vessels from non-heartbeating donors in general and subsequent prolonged cold storage seems feasible when vessels are recovered within 2 hours. The use of antibiotics needs to be carefully assessed for each intended-to-use tissue. For vessels tested, a combination of gentamycin, piperacillin, and metronidazole supported the maintenance of vessel function.