The percutaneous muscle biopsy procedure is an invaluable tool for characterizing skeletal muscle and capillarization. Little is known about methodological or biological variation stemming from the technique in heterogeneous muscle. Five muscle biopsies were taken from the vastus lateralis of a group of young men (n = 29, 22 ± 1 years) over a 96-h period. We investigated the repeatability of fibre distribution, indices of muscle capillarization and perfusion, and myofibre characteristics. No differences between the biopsies were reported in myofibre type distribution, cross-sectional area (CSA), and perimeter. Capillary-to-fibre perimeter exchange index and individual capillary-fibre contacts were unchanged with respect to the location of the muscle biopsy and index of capillarization. The variability in the sampling distribution of fibre type specific muscle CSA increased when fewer than 150 muscle fibres were quantified. Variability in fibre type distribution increased when fewer than 150 muscle fibres were quantified. Myofibre characteristics and indices of capillarization are largely consistent throughout the vastus lateralis when assessed via the skeletal muscle biopsy technique.

Novelty

• Markers of muscle capillarization and perfusion were unchanged across multiple sites of the human vastus lateralis.
• Myofibre characteristics such as muscle cross-sectional area, perimeter, and fibre type distribution were also unchanged.
• Variation of muscle CSA was higher when fewer than 150 muscle fibres were quantified.