Intra- and inter-organ signalling are critical for normal function, contribute to disease progression, and are processes in which extracellular vesicles (EVs) are emerging as fundamental mediating entities. However, our current understanding of cardiac EVs (cEVs) has been acquired from in vitro culture systems, a limitation resultant of challenges with tissue-EV extraction. Here, we present a novel strategy to isolate cEVs using gentle enzymatic perfusion of an intact heart. cEVs are 100-300 nm in diameter, attainable at levels of 2-3µg protein/heart, and carry many classical EV markers associated with both surface and endosomal origin. Proteomic dissection of cEVs highlights enrichment of components associated with cardiac regulatory functions, including signalling, peptide-hormone processing, and extracellular matrix maintenance, as well proteins enriched in organs (i.e., liver, kidney) other than the heart. This unique insight into cEVs directly from heart provides the basis for further investigation into cEVs and how cardiovascular diseases dysregulate their form and function in intra-cardiac and inter-organ communication.