Prolonged ischemia of skeletal muscle tissue, followed by reperfusion, leads to ischemia/reperfusion injury (IRI), which is a feared local and systemic inflammatory reaction. With respect to the 3Rs, we wanted to determine which parameters for assessment of IRI require a reperfusion time of 24 h and for which 2¿h of reperfusion are sufficient. Rats were subjected to 3¿h of hind limb ischemia and 2¿h or 24¿h of reperfusion. Human plasma derived C1 inhibitor was used as a drug to prevent reperfusion injury. For 2¿h of reperfusion the rats stayed under anesthesia throughout (severity grade 1), whereas for 24¿h they were awake under analgesia during reperfusion (grade 2). The femoral artery was clamped and a tourniquet was placed, under maintenance of venous return. C1 esterase inhibitor was systemically administered 5¿min before the induction of ischemia. No differences in local muscle edema formation and depositions of immunoglobulin G and immunoglobulin M were observed between 2¿h and 24¿h (P¿>¿0.05), whereas lung edema was only observed after 24¿h. Muscle viability was significantly lower after 24¿h vs 2¿h reperfusion (P¿<¿0.05). Increased plasma creatine kinase (CK)-MM and platelet-derived growth factor (PDGF)-bb could be detected after 2¿h, but not after 24¿h of reperfusion. By contrast, depositions of C3b/c and fibrin in muscle were only detected after 24¿h (P¿<¿0.001). In conclusion, for a first screening of drug candidates to reduce IRI, 2¿h reperfusions are sufficient, and these reduce the severity of the animal experiment. Twenty-four-hour reperfusions are only needed for in-depth analysis of the mechanisms of IRI, including lung damage.