Exercise training can increase both mitochondrial content and mitochondrial respiration. Despite its popularity, high-intensity exercise can be accompanied by mild acidosis (also present in certain pathological states), which may limit exercise-induced adaptations to skeletal muscle mitochondria. The aim of this study was to determine if administration of ammonium chloride (0.05 g/kg) to Wistar rats before each individual exercise session (5 high-intensity exercise sessions per week for eight weeks) reduced training-induced increases in mitochondrial content (measured by citrate synthase activity and protein content of electron transport system complexes) and respiration (measured in permeabilised muscle fibres). In the soleus muscle, the exercise-training-induced increase in mitochondrial respiration was reduced in rats administered ammonium chloride compared to control animals, but mitochondrial content was not altered. These effects were not present in the white gastrocnemius muscle. In conclusion, ammonium chloride administration before each exercise session over eight weeks reduced improvements in mitochondrial respiration in the soleus muscle but did not alter mitochondrial content. This suggests that mild acidosis may impact training-induced improvements in the respiration of mitochondria in some muscles.