Exercise is considered to be beneficial for free fatty acid (FFA) metabolism, although the effects of increased physical activity on FFA uptake and oxidation in different tissues in vivo in humans have been inconsistent. To investigate the heredity-independent effects of physical activity and fitness on FFA uptake in skeletal muscle, myocardium, and liver we used positron emission tomography (PET) in nine healthy young male monozygotic twin pairs discordant for physical activity and fitness. The co-twins with higher physical activity constituting the more active group had a similar body mass index but less body fat and 18±10% higher VO₂max (p<0.001) compared to the less active brothers with lower physical activity. Low-intensity knee-extension exercise increased skeletal muscle FFA and oxygen uptake six to ten times compared to resting values but no differences were observed between the groups at rest or during exercise. At rest the more active group had lower hepatic FFA uptake compared to the less active group (5.5±4.3 vs. 9.0±6.1 µmol•100ml^-1•min^-1, p=0.04). Hepatic FFA uptake associated significantly with body fat percentage (p=0.05). Myocardial FFA uptake was similar between the groups. In conclusion, in the absence of the confounding effects of genetic factors, moderately increased physical activity and aerobic fitness decrease body adiposity even in normal-weighted healthy young adult men. Further, increased physical activity together with decreased intra-abdominal adiposity seems to decrease hepatic FFA uptake but has no effects on skeletal muscle or myocardial FFA uptake.