Remote ischaemic preconditioning (RIPC) is a therapeutic intervention that has been demonstrated to reduce myocardial injury in the clinical setting. However, the underlying cardioprotective mechanisms remain unclear. We hypothesised that RIPC utilises both humoral and neural pathways to convey the cardioprotective signal from the preconditioned remote organ to the heart. C57BL/6 mice were anaesthetised and subjected to in vivo 30 min coronary artery ischaemia followed by 120 min of myocardial reperfusion, at the end of which myocardial infarct size was measured and expressed as a percentage of the risk zone. RIPC was induced by 3 cycles of 5 min left femoral artery occlusion interspersed with 5 min reperfusion before prolonged myocardial ischaemia with or without femoral vein occlusion (humoral pathway), femoral nerve resection and/or sciatic nerve resection (neural pathway). RIPC resulted in a smaller myocardial infarct size when compared to control. However, occluding the femoral vein completely abolished the infarct-limiting effect of RIPC. Similarly, combined femoral and sciatic nerve resection also abolished the cardioprotective effect of RIPC. Interestingly, femoral nerve or sciatic nerve resection alone only partially abolished the infarct-limiting effect of RIPC. In conclusion, remote limb ischaemic preconditioning reduced myocardial infarct size in the mice in a manner which implicates both a neural and humoral pathway.