Aim Mitochondria undergo continuous changes in shape as result of complex fusion and fission processes. The physiological relevance of mitochondrial dynamics is still unclear. In the field of mitochondria bioenergetics, there is a need of tools to assess cell mitochondrial content. To develop a method to visualize mitochondrial networks in high resolution and assess mitochondrial volume. Methods Confocal fluorescence microscopy imaging of mitochondrial network stains in human vastus lateralis single muscle fibres and focused ion beam/ scanning electron microscopy (FIB/SEM) imaging, combined with 3D reconstruction was used as a tool to analyse mitochondrial morphology and measure mitochondrial fractional volume. Results Most type I and type II muscle fibres have tubular highly interconnected profusion mitochondria, which are thicker and more structured in type I muscle fibres (Fig. 1). In some muscle fibres, profission‐isolated ellipsoid‐shaped mitochondria were observed. Mitochondrial volume was significantly higher in type I muscle fibres and showed no correlation with any of the investigated molecular and biochemical mitochondrial measurements (Fig. 2). Three‐dimensional reconstruction of FIB/SEM data sets shows that some subsarcolemmal mitochondria are physically interconnected with some intermyofibrillar mitochondria (Fig. 3). Conclusion Two microscopy methods to visualize skeletal muscle mitochondrial networks in 3D are described and can be used as tools to investigate mitochondrial dynamics in response to life‐style interventions and/or in certain pathologies. Our results question the classification of mitochondria into subsarcolemmal and intermyofibrillar pools, as they are physically interconnected.