Mitochondria play a central role in cellular metabolism and are crucial for maintaining cellular health. Exercise is a proven intervention to enhance mitochondrial function and prevent non-communicable diseases, yet the molecular mechanisms driving exercise-induced mitochondrial remodelling remain unclear. This study compares the effects of moderate-intensity continuous exercise (MICE) and sprint-interval exercise (SIE) on mitochondrial adaptations. We demonstrate that SIE induces mitochondrial stress and activates the mitochondrial unfolded protein response (UPRmt), alongside disturbances in mitochondrial structure while MICE promotes mitochondrial adaptations characterized by increased mitochondrial content. Over an 8-week training period, moderate-intensity continuous training (MICT) and sprint-interval training (SIT) show divergent mitochondrial remodelling. Findings provide novel insights into the mechanisms by which different exercise prescriptions modulate mitochondrial signalling and remodelling in human skeletal muscle, offering a basis for personalized exercise strategies to optimize mitochondrial health.