The skeletal muscle interstitial space is the extracellular region around myofibres and mediates crosstalk between resident cell types. We applied a proteomic workflow to characterise the human skeletal muscle interstitial fluid proteome at rest and in response to exercise. Following exhaustive exercise, markers of skeletal muscle damage accumulate in the interstitial space followed in turn by the appearance of immune cell-derived proteins. Among the proteins upregulated after exercise, we identified cathelicidin-related antimicrobial peptide (CAMP) as a bioactive molecule regulating muscle fibre development. Treatment with the bioactive peptide derivative of CAMP (LL-37) resulted in the growth of larger C2C12 skeletal muscle myotubes. Phosphoproteomics revealed that LL-37 activated pathways central to muscle growth and proliferation, including PI3K, AKT, MAPKs, and mTOR. Our findings provide a proof-of-concept that the interstitial fluid proteome can be quantified via microdialysis sampling in vivo. Furthermore, these data highlight the importance of cellular communication in the adaptive response to exercise.