The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (including exosomes) involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24 h rhythmic abundance of individual proteins in exosomes using liquid chromatography-mass spectrometry in circadian-synchronised tendon fibroblasts. Further, the release of exosomes enriched in RNA-binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Finally, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast exosomes is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for exosomes released by tendon fibroblasts which supports the view that the circadian clock regulates protein cargo in exosomes involved in cell-cell crosstalk.