Permeabilized muscle fibers (pfi) are widely used to assess mitochondrial (mt) respiratory function in skeletal muscle of various models in different physiological and pathological conditions. Facing the numerous data available for mt-respiration from the literature, it remains challenging to determine what the right values are for a specific respiratory protocol. Moreover, mt-respiration values are highly dependent on pfi preparation, which required good technical skills. In the frame of COST Action MITOEAGLE, one of the objectives of WG2 is the generation of reference values for mitochondrial respirometry in permeabilized skeletal muscle sample preparations. The idea is that new researchers in the field follow a reference protocol and check if their values are in an acceptable range. This approach could serve to test researchers’ technical skills and therefore determine if they are proficient enough to perform their own experiments with confidence.