The activity and quantity of mitochondrial proteins and the mitochondrial volume density (MitoVD) are higher in trained muscles; however, the underlying mechanisms remain unclear. Our goal was to determine if 20 weeks’ endurance training simultaneously increases running performance, the amount and activity of mitochondrial proteins, and MitoVD in the gastrocnemius muscle in humans. Eight healthy, untrained young men completed a 20-week moderate-intensity running training program. The training increased the mean speed of a 1500 m run by 14.0% (p = 0.008) and the running speed at 85% of maximal heart rate by 9.6% (p = 0.008). In the gastrocnemius muscle, training significantly increased mitochondrial dynamics markers, i.e., peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) by 23%, mitochondrial transcription factor A (TFAM) by 29%, optic artrophy-1 (OPA1) by 31% and mitochondrial fission factor (MFF) by 44%, and voltage-dependent anion channel 1 (VDAC1) by 30%. Furthermore, training increased the amount and maximal activity of citrate synthase (CS) by 10% and 65%, respectively, and the amount and maximal activity of cytochrome c oxidase (COX) by 57% and 42%, respectively, but had no effect on the total MitoVD in the gastrocnemius muscle. We concluded that not MitoVD per se, but mitochondrial COX activity (reflecting oxidative phosphorylation activity), should be regarded as a biomarker of muscle adaptation to endurance training in beginner runners.