The number of studies on mitochondrial function is growing as a result of the recognition of the pivotal 24 role of an intact mitochondrial function in numerous diseases. Measurements of oxygen consumption by 25 the mitochondria in human skeletal muscle are used in many studies. There are several advantages of 26 studying mitochondrial respiration in permeabilized fibers (Pfi), but the method requires a manual 27 procedure of mechanical separation of the fiber bundles in the biopsy and chemical permeabilization 28 Q1 of the cell membrane. This is time-consuming and subject to interpersonal variability. An alternative is 29 to use a semiautomatic tool for preparation of a homogenate of the muscle biopsy. We investigated 30 whether the PBI shredder is useful in preparing amuscle homogenate for measurements of mitochondrial 31 respiratory capacity. The homogenate is compared with the Pfi preparation. Maximal respiratory capacity 32 was significantly reduced in the homogenate compared with the Pfi from human skeletal muscle. A 33 marked cytochrome c response was observed in the homogenate, which was not the case with the Pfi, 34 indicating that the outer mitochondrial membrane was not intact. The mitochondria in the homogenate 35 were more uncoupled compared with the Pfi. Manual permeabilization is an advantageous technique for 36 preparing human skeletal muscle biopsies for respirometry.