Introduction: It is hypothesized, that increased production of reactive oxygen species (ROS) in endothelial cells may contribute to the development of endothelial dysfunction in cardiovascular disease. In order to test this, we have developed a novel method to determine mitochondrial respiration and ROS production in a limited number of endothelial cells isolated from skeletal muscle samples of healthy humans and rats and, for comparison, human umbilical vein endothelial cells (HUVEC). Method: Muscle endothelial cells were isolated from muscle samples obtained from the thigh of healthy young subjects and the lower limb of rats by use of antibody coated magnetic beads. The H2O2 emission (ROS) was measured fluorometric in intact endothelial cells with an Oxygraph (Oxygraph-2K, Oroboros Instruments). The method allows for measuring real time oxygen consumption using a respirometer, and ROS production is measured fluorometric with an add-on module to the Oxygraph for the simultaneous measurement of respiration and H2O2 production of approximately 100.000 cells in the chamber. High (11mM) compared to normal physiological (5mM) glucose levels were acutely incubated with measurement in the Oxygraph in HUVEC to simulate the effect of high glucose levels in the bloodstream. Results: The isolation procedure of cells resulted in over 1 million cells per preparation from the rat tissue and the human muscle biopsies. The oxygen consumption tended to be higher in endothelial cells from the microvascular system in rat muscle compared to HUVEC at baseline level (88±16 vs. 24±4 pmol O2/S×106 cells; p=0.060; n=4). Oxygen consumption in human microvascular endothelial cells was 75% (p<0.001; n=4) lower than in microvascular endothelial cells from rat. No difference in oxygen consumption found between human microvascular endothelial cells and HUVEC. However, the ROS production was not different between the cell types. The oxygen consumption was 12% lower (9±2 vs. 10±2 pmol O2/S×106 cells; n=2) in acutely incubated high glucose compared to normal levels of glucose in HUVEC. The ROS production was 7% higher (0.37±0.03 vs. 0.35±0.01 pmol H2O2/S×106 cells; n=2) in high compared to normal levels of glucose. Despite the oxygen consumption was lowered in the cells incubated in high glucose, the ROS production seems to be higher from the preliminary data. These preliminary data is very important for understanding, how the environment affects the endothelial mitochondria. Furthermore, understanding how mitochondrial function may influence cell function will provide important novel information regarding cardiovascular disease. Conclusion: Our data show, that it is possible to isolate sufficient number of endothelial cells from skeletal muscle biopsy samples for analysis of mitochondrial respiration and ROS formation. The procedure should provide a useful in vitro model for studying various aspects of microvascular biology and pathology.