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. Aim: 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 fibers and, focused ion beam scanning electron microscopy (FIB/SEM) imaging, combined with 3D reconstruction was used as a tool to analyze mitochondrial morphology and measure mitochondrial fractional volume. Results: Most type I and type II muscle fibers have tubular highly interconnected profusion mitochondria, which are thicker and more structured in type I muscle fibers (Figure 1). In some muscle fibers, profission isolated ellipsoid-shaped mitochondria were observed. Mitochondrial volume was significantly higher in type I muscle fibers and showed no correlation with any of the investigated molecular and biochemical mitochondrial measurements (Figure 2). Three dimensional reconstruction of FIB/SEM data sets shows that some subsarcolemmal mitochondria are physically interconnected with some intermyofibrillar mitochondria (Figure 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, since they are physically interconnected. This article is protected by copyright. All rights reserved.