Microbiopsies are increasingly used as an alternative to the standard Bergstrom technique for skeletal muscle sampling. The potential impact of these two different procedures on mitochondrial respiration rate is unknown. The objective of this work was to compare microbiopsies versus Bergstrom procedure on mitochondrial respiration in skeletal muscle. 52 vastus lateralis muscle samples were obtained from 13 anesthetized pigs, either with a Bergstrom [6 gauges (G)] needle or with microbiopsy needles (12, 14, 18G). Maximal mitochondrial respiration (V (GM-ADP)) was assessed using an oxygraphic method on permeabilized fibers. The weight of the muscle samples and V (GM-ADP) decreased with the increasing gauge of the needles. A positive nonlinear relationship was observed between the weight of the muscle sample and the level of maximal mitochondrial respiration (r = 0.99, p < 0.05) and between needle size and maximal mitochondrial respiration (r = 0.99, p < 0.05). Microbiopsies give lower muscle sample weight and maximal rate of mitochondrial respiration compared to the standard Bergstrom needle.Therefore, the higher the gauge (i.e. the smaller the size) of the microbiopsy needle, the lower is the maximal rate of respiration. Microbiopsies of skeletal muscle underestimate the maximal mitochondrial respiration rate, and this finding needs to be highlighted for adequate interpretation and comparison with literature data.