Key points It is generally accepted that mitochondrial volume density in human skeletal muscle diminishes with chronic high altitude exposure. All data supporting this concept were collected during mountaineering expeditions, which are associated with the confounding effects of whole body negative energy balance. Here we examine the effect of 28 days of exposure to 3454 m on skeletal muscle mitochondrial volume density in a setting where whole body weight, whole body composition, leg lean mass, skeletal muscle fibre area and maximal power output were preserved. Our results demonstrate that total skeletal muscle mitochondrial volume density increases in response to high altitude exposure secondary to a preferential increase in intermyofibrillar mitochondrial populations. This study provides direct evidence contradicting the notion that high altitude exposure diminishes skeletal muscle mitochondrial volume density, highlighting an inconsistent understanding of the role of hypoxia on skeletal muscle mitochondria. Abstract The role of hypoxia on skeletal muscle mitochondria is controversial. Studies superimposing exercise training on hypoxic exposure demonstrate an increase in skeletal muscle mitochondrial volume density (MitoVD) over equivalent normoxic training. In contrast, reductions in both skeletal muscle mass and MitoVD have been reported following mountaineering expeditions. These observations may, however, be confounded by negative energy balance, which may obscure the results. Accordingly we sought to examine the effects of high altitude hypoxic exposure on mitochondrial characteristics, with emphasis on MitoVD, while minimizing changes in energy balance. For this purpose, skeletal muscle biopsies were obtained from nine lowlanders at sea level (Pre) and following 7 and 28 days of exposure to 3454 m. Maximal ergometer power output, whole body weight and composition, leg lean mass and skeletal muscle fibre area all remained unchanged following the altitude exposure. Transmission electron microscopy determined that intermyofibrillar (IMF) MitoVD was augmented (P = 0.028) by 11.5 ± 9.2% from Pre (5.05 ± 0.9%) to 28 Days (5.61 ± 0.04%). In contrast, there was no change in subsarcolemmal (SS) MitoVD. As a result, total MitoVD (IMF + SS) was increased (P = 0.031) from 6.20 ± 1.5% at Pre to 6.62 ± 1.4% at 28 Days (7.8 ± 9.3%). At the same time no changes in mass‐specific respiratory capacities, mitochondrial protein or antioxidant content were found. This study demonstrates that skeletal muscle MitoVD may increase with 28 days acclimation to 3454 m.