Objectives Aerobic exercise training can increase skeletal muscle mitochondrial content. Supine exercise training with legs above the heart potentially augments these increases. However, the impact of supine exercise training on mitochondrial biogenesis and cardiovascular adaptations remains unclear. Methods In this single-centred, randomized, parallel arm trial, 19 recreationally active individuals underwent seven sessions of either supine with legs up (SUP; n=9, 6 females) or upright with legs down (UP; n=10, 7 females) aerobic training on a recumbent bike at 71 ± 7 % and 71 ± 2 % of peak work rate (WRpeak), respectively. The study aimed to test the effects of training with decreased muscle oxygenation on indices of muscle mitochondrial remodelling. Secondary outcomes included exercise performance, muscle oxygenation, and cardiovascular responses. Results Secondary outcomes revealed significant interaction effects for time to fatigue (TTF) and WRpeak in the SUP group during supine testing, suggesting enhanced exercise tolerance and performance. No between group interaction effects were observed for upright testing. No clear effects on mitochondrial biogenesis were observed based on expression of mitochondrial protein subunits and transcriptional regulators. Acutely, HRpeak was lower during the SUP Test compared to the UP Test. No central cardiovascular adaptations were observed following training. Conclusions Our exploratory analyses showed that supine aerobic training more effectively improves supine exercise tolerance and performance compared with upright training, despite no differences in measured proteins related to mitochondrial biogenesis. Further research is needed to elucidate the mechanisms underlying these postural-specific training effects.