In addition to generating the bulk of cellular energy, mitochondria direct a vast array of biological functions essential for cellular homeostasis. Mutations affecting mitochondrial function and biogenesis cause mitochondrial diseases which affect tissues of high energy demand such as heart, skeletal muscle and brain. Mitochondrial dysfunction has also been implicated in various cancers and aging. A long-standing question in biology concerns the biogenesis of mitochondria and its regulation in response to stress and the metabolic needs of the cellular environment. Exercise represents a major challenge to both these pathways while also being arguably one of the most ‘natural’ perturbations available. Despite this, there has been little study into exactly how mitochondria adapt to variable exercise conditions. In order to further demonstrate the effects exercise has on the mitochondria, ten participants underwent three different training volume phases over 12 weeks. Tissue biopsies were taken prior to commencing the study and after each phase and then subjected to a panel of bioenergetic assessments to measure oxidative capacity. Mitochondria were isolated from muscle biopsy material and their proteomes analysed by label-free quantitative mass-spectrometry. Training phases included a combination of normal-, high- and reduced-volume training regimens. By incorporating a combination of variable volumes of exercise we illustrated the effects these training volumes have on mitochondrial biogenesis, energetic capacity and the mitochondrial proteome more broadly. We observed extensive mitochondrial biogenesis in response to changing volumes of exercise training. While this was met with an overall increase in oxidative capacity, mitochondria underwent extensive remodelling of energetic pathways. Cessation of high-volume exercise reversed some, but not all of these changes. Our findings suggest that training volume is an important determinant of changes in mitochondrial content and function and is a useful model to help to further our understanding of the fundamental mechanisms of mitochondrial biogenesis.