Cardiac function is dynamically regulated by intercellular signalling. As an important intercellular signalling mediator, this study focused on isolating and defining membrane bound signalling packages termed extracellular vesicles (EVs) directly from heart. Here, we utilised a gentle, enzymatic perfusion to extract EVs from whole cardiac tissues (cEVs) for biophysical and proteomic characterisation; cEVs (100-300 nm) are Cd63+, Tsg101+, Pdcd6ip/Alix+ (EV markers) and contain proteins associated with cardiomyocyte (Actn1, Myh6), cardiac progenitor (Cd34, Abcg2), smooth muscle (Cald1, Tagln), endothelial (Vwf), and fibroblast (Ckap4) origins. Proteomic profiling of cEVs revealed 1721 proteins, which bioinformatics revealed hold an enrichment in metabolic functions of glycolysis, fatty acid oxidation, and antioxidant action, presumably to meet the high energetic demands and balance resulting oxidative stress in cardiac tissue.