Exercise promotes health through adaptive metabolic and mechanical remodelling, mediated by a network of kinases in response to homeostatic stress. Identifying the specific signalling changes that drive beneficial adaptations in exercise would provide novel drug targets for a range of diseases. Multiple stress- and metabolic-sensing pathways are acutely activated during exercise, however it is unclear whether they interact. We hypothesised that combining exercise-like treatments could recapitulate signalling interactions in vitro. Since interactions may contribute to beneficial effects of exercise, interrogating these effects rather than simple linear pathways is crucial to understanding exercise response. Comprehensive screening of exercise-like treatments in rat L6 myotubes ranked isoproterenol and thapsigargin as the most similar to the in vivo human skeletal muscle acute exercise phosphoproteome. Measuring global phosphoproteomes of L6 myotubes stimulated with thapsigargin, isoproterenol and their combination quantified >20,000 Class I phosphopeptides, of which 25% were regulated in at least one treatment. Strikingly, the combination of isoproterenol and thapsigargin uniquely regulated 962 phosphosites that were not regulated in either treatment alone. These unique sites were highly correlated to those regulated in exercised human biopsies suggesting that interactions recapitulate in vivo exercise signal transduction. Exercise regulates the secretion of factors to produce health benefits throughout the body. However, the underlying mechanisms regulating secretion are poorly understood. To investigate this, we measured the secretome of exercise-like treatments to delineate the pathways controlling protein secretion. Remarkably, the combined exercise-like treatments uniquely modulated the secretion of 80 proteins. Most notably, the secretion of fibrosis and thrombosis-stimulating proteins such as Serpine1 were downregulated by these treatments. Regulated phosphosites appear to drive this phenotype, including multiple phosphosites on the Serpine1 mRNA degrading protein Serbp1. This work provides a resource to link exercise-related signalling changes to protein secretion and other exercise-related phenotypes.