Evaluating the activity of cardiac mitochondria is probably the best way to estimate early cellular damage in chronic pathology. Early diagnosis allows rapid therapeutic intervention thus increasing patient survival rate in a number of diseases. However, data on human cardiac mitochondria are scarce in the international literature. Here, we describe a method to extract and study functional mitochondria from the small-sized right atrial aliquots (minimum of 400 mg) obtained during extracorporeal circulation and usually considered as surgical waste products. The mitochondria were purified through several mechanical processes (fine myocardial cutting, tissue grinding and potter Elvehjem homogenising), an enzymatic proteolytic action (subtilisin) and differential centrifugations. In chronic pathologies, including obesity, early disturbances of mitochondrial function can occur. The effects of obesity on the rate of mitochondrial oxygen consumption and H2O2 release were thus determined with three different substrates (glutamate/malate, succinate/rotenone and palmitoylcarnitine/malate). The human atrial mitochondria were of high quality from a functional viewpoint, compared to rat ventricle organelles, but the extraction yield of the human mitochondria was twice lower than that of rat mitochondria. Tests showed that glutamate/malate-related ADP-stimulated respiration was strongly increased in obese subjects, although the oxidation of the other two substrates was unaffected. Reactive oxygen species (ROS) production by the isolated mitochondria was low in comparison with that of the lean subjects. These results confirm those found in one of our previous studies in the ventricles of rats fed a high-fat diet. In conclusion, the described method is simple, reliable and sensitive. It allows for the description of the impact of obesity on the function of atrial mitochondria while using only a small patient sampling (n = 5 in both the lean and the obese groups).