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OBJECTIVES The objectives were to investigate 1) the effect of acute aerobic exercise on tumour hypoxia and blood perfusion, 2) the impact of exercise intensity, 3) the duration of the effect, and 4) the effect of prolonged training on tumour hypoxia as well as tumour growth. METHODS Female CDF1 mice were inoculated with the C3H mammary carcinoma either in the mammary fat pad or subcutaneously in the back. For experiments on the effect of different intensities in a single exercise bout, mice were randomized to 30 min treadmill running low, moderate, or high intensity speeds or no exercise. To investigate the prolonged effect on hypoxia and tumour growth, tumour-bearing mice were randomized to no exercise (CON) or daily 30 min high-intensity exercise averaging two weeks (EX). Tumour hypoxic fraction was quantified using the hypoxia marker Pimonidazole. RESULTS Initially, high intensity exercise reduced tumour hypoxic fraction by 37% compared with CON (p=0.046; 95% CI: 0.1; 10.3) in fat pad tumours. Low and moderate intensities did not. Following experiments investigating the duration of the effect - as well as experiments in mice with back tumours - failed to show any exercise induced changes in hypoxia. Interestingly, prolonged daily training significantly reduced hypoxic fraction by 60% (p=0.002; 95% CI: 2.5;10.1) compared with CON. CONCLUSION Despite diverging findings on the acute effect of exercise on hypoxia, our data indicate that if exercise has a diminishing effect, high-intensity exercise is needed. Prolonged training reduced tumour hypoxic fraction - cautiously suggesting a potential clinical potential.