The plasma peptidome contains the entire complement of low molecular weight endogenous peptides derived from secretion, protease activity and post-translational modifications and is a rich source of biomarkers and novel bioactives. To determine the utility of peptidomics we examined the effects of exercise on the plasma peptidome because it conveys many of its physiological benefits via the blood stream. We compared methods to rapidly isolate the plasma peptidome revealing trichloroacetic acetic precipitation under denaturing conditions combined with mixed-mode solid-phase extraction produces the greatest depth. We applied this approach to the multiplexed temporal quantification of the exercise-regulated plasma peptidome combined with 2D-LC-MS/MS. Peptides were analysed with multiple fragmentation methods including HCD and EThcD which resulted in the quantification of 5,548 unique endogenous peptides. The plasma peptidome underwent dynamic changes during exercise on a time-scale of minutes and this was rapidly reversible following exercise cessation. Among acutely regulated peptides were many known hormones including insulin, glucagon, ghrelin, bradykinin, cholecystokinin and secretogranins. Using site-specific protease mapping to known substrates and subsequent enrichment analysis, we generated a protease:substrate network to infer the activity of proteases regulated with exercise. Our analysis also included the characterisation of PTMs such as amidation and glycosylation. The utility of multiple fragmentation methods were able localise N-glycan modification sites in the glycopeptidome. These data reveal the utility of peptidomic quantification to temporally quantify bioactive peptides, biomarkers, PTMs and proteolytic activity during pathophysiological processes.