AbstractSustained ryanodine receptor (RyR) Ca²⁺ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca²⁺ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca²⁺ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca²⁺ leak or preventing mitochondrial Ca²⁺ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca²⁺ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise.