The aim of this study was to verify the possibility of preparation and effectiveness of the use of blood plasma containing an effector of ischemic tolerance activated by applying two sublethal stresses to a donor. As sublethal stresses, two periods of 20-minute hindlimb ischemia were used with a two-day interval between them. Active plasma was isolated six hours after the second hindlimb ischemia. The effectiveness of active plasma as well as remote postconditioning was tested after three hours of tourniquet-induced ischemia on the gastrocnemius muscle. The wet/dry ratio of gastrocnemius muscle (degree of tissue oedema), nitroblue tetrazolium reduction (tissue necrosis), and CatWalk test (hind limb functionality) were evaluated 24 h after the end of ischemia. Three hours of ischemia increased muscle oedema and necrosis in comparison to control by 26.72% (p < 0.001) and 41.58% (p < 0.001) respectively. Remote ischemic postconditioning as well as injection of conditioned blood plasma significantly prevented these changes, even when they were applied one or three hours after the end of ischemia. Equally effective double-conditioned plasma appears to have better prospects in life-threatening situations such as stroke and myocardial infarction.