In response to exercise, networks of protein kinases and signalling pathways become rapidly engaged to blunt the homeostatic threats generated by contraction-induced increases in skeletal muscle energy and oxygen demand. Skeletal muscle AMP-activated protein kinase (AMPK) serves important physiological roles in humans and rodents via sensing cellular energy status and mobilising key energy reserves such as glycogen in response to high-intensity exercise. AMPK has the capacity to bind glycogen via its β subunit’s carbohydrate binding module. While exercise commenced with reduced skeletal muscle glycogen is known to amplify skeletal muscle exercise-regulated signalling pathways such as AMPK, the physiological and metabolic consequences of disrupting AMPK-glycogen interactions in vivo remain incompletely understood. Furthermore, while high-intensity interval training (HIIT)-based exercise stimulates skeletal muscle glycogen utilisation and elicits similar or superior health-promoting physiological adaptations versus moderate-intensity continuous training (MICT), the breadth of exercise modality-specific signalling pathways underlying HIIT remain unknown. Using physiological, phosphoproteomic and metabolomic approaches, our research group has uncovered the human skeletal muscle exercise signalling responses to an acute bout of HIIT versus workload- and duration-matched MICT, as well as mapped the skeletal muscle, liver and plasma metabolomic responses to acute exercise in wild type versus AMPK β subunit knock-in mice with disrupted AMPK-glycogen binding capacity. These studies have revealed unique signalling pathways regulated by HIIT in human skeletal muscle and metabolic pathways underlying the phenotypic effects of disrupting AMPK-glycogen interactions in mice. Collectively, these studies lay the foundation for future investigation of AMPK- and exercise-regulated signalling pathways and metabolic regulatory mechanisms involved in maintaining energy homeostasis and eliciting the beneficial effects of exercise.