Background <p>Maximal oxygen uptake (VO_2max) is the gold standard for assessing cardiopulmonary fitness. However, the link between VO_2max and mitochondrial function is complex, with no direct causality proven. Analysing mtDNA copy number offers an innovative way to understand aerobic performance by measuring mitochondrial biogenesis, although its relation to physical performance and fitness is not well studied, especially in trained individuals.</p> Objective <p>This study aimed to compare VO_2max and mtDNA copy number in blood leukocytes between highly trained runners and nontrained men. It also examined how leukocyte mtDNA content adapts to a 6-week HIIT aerobic training programme in nontrained individuals.</p> Methods <p>We conducted a cross-sectional study with 20 highly trained runners and 20 nontrained healthy subjects. Then, we implemented a 6-week HIIT training programme for the nontrained group, comparing their VO_2max and mtDNA copy number to a control group. Participants trained 4&#xa0;min/4&#xa0;min HIIT treadmill running, three times a week.</p> Results <p>The cross-sectional part showed that highly trained runners had significantly higher mtDNA copy numbers than nontrained subjects (<i>p</i> = 0.046; <i>d</i> = 0.652; 95% CI: [0.01, 1.28]; [medium–large effect]); further, we observed a significant positive correlation between mtDNA copy number and VO_2max combining both groups (<i>p</i> = 0.013; R² = 0.153 [small–moderate effect]). After the 6-week HIIT programme, the intervention group showed significant increases in both VO_2max (<i>p</i> &lt; 0.001) and mtDNA copy number (<i>p</i> = 0.015), with large and medium effect sizes, respectively. The intervention group’s mtDNA copy number increased by 321.6 ± 391.6%; 95% CI: [120.2, 522.9], compared to 12.8 ± 32.8%; 95% CI: [−&#xa0;5.4, 30.9] in controls, indicating substantial interindividual variability, which could be attributed to a combination of biological factors, but we also observed indications of a potential responder versus non-responder pattern.</p> Conclusion <p>These findings align with previous research, indicating that mtDNA copy number correlates with VO_2max, being higher in highly trained runners than in nontrained subjects, and increasing with training after a 6-week HIIT intervention. This study is novel in showing that a 6-week HIIT programme significantly boosts leukocyte mtDNA content, enhancing our understanding of individual adaptations to endurance training.</p>