Aims We examined coupling of myocardial ischaemic tolerance to physical activity and inactivity, and whether this involves modulation of survival (AKT, AMPK, ERK1/2, HSP27, EGFR) and injury (GSK3β) proteins implicated in ischaemic preconditioning and calorie restriction. Methods Proteomic modifications were assessed in ventricular myocardium, and tolerance to 25‐min ischaemia in ex vivo perfused hearts from C57Bl/6 mice subjected to 14‐day voluntary activity in running‐naïve animals (Active); 7 days of subsequent inactivity (Inactive); brief (day 3) restoration of running (Re‐Active); or time‐matched inactivity. Results Active mice increased running speed and distance by 75–150% over 14 days (to ~40 m min−1 and 10 km day−1), with Active hearts resistant to post‐ischaemic dysfunction (40–50% improvements in ventricular pressure development, diastolic pressure and dP/dt). Cardioprotection was accompanied by ~twofold elevations in AKT, AMPK, HSP27 and GSK3β phosphorylation and EGFR expression. Ischaemic tolerance was reversed in Inactive hearts, paralleling reduced EGFR expression and GSK3β and ERK1/2 phosphorylation (AKT, AMPK, HSP27 phosphorylation unaltered). Running characteristics, ischaemic tolerance, EGFR expression and GSK3β phosphorylation returned to Active levels within 1–3 days of restored activity (without changes in AKT, AMPK or HSP27 phosphorylation). Transcriptional responses included activity‐dependent Anp induction vs. Hmox1 and Sirt3 suppression, and inactivity‐dependent Adora2b induction. Conclusions Data confirm the sensitive coupling of ischaemic tolerance to activity: voluntary running induces cardioprotection that dissipates within 1 week of inactivity yet recovers rapidly upon subsequent activity. While exercise in naïve animals induces a molecular profile characteristic of preconditioning/calorie restriction, only GSK3β and EGFR modulation consistently parallel activity‐ and inactivity‐dependent ischaemic tolerance.