We investigated heredity-independent effects of increased physical activity and aerobic fitness on skeletal muscle free fatty acid (FFA) uptake, perfusion, and their heterogeneity at rest and during exercise. Also the relationship between local skeletal muscle FFA uptake and perfusion were studied. Nine young adult male monozygotic twin pairs with significant difference in physical activity (229±156 min average time spent for conditioning exercise per week in more and 98±71 min in less active twins, p=0.013) and aerobic fitness (18±10% difference in VO_2max) between brothers were studied using positron emission tomography. Submaximal knee-extension exercise increased perfusion, FFA uptake, and oxygen uptake in quadriceps femoris muscles six to ten times compared to resting values (p<0.001). More active twins tended to utilize more oxygen while no differences were found in muscle perfusion or FFA uptake between groups. Mean perfusion and FFA uptake correlated strongly at a whole muscle level both at rest (r=0.97, p=0.03 in more and r=0.98, p=0.02 in less active twins) and during exercise (r=0.99, p=0.01 and r=0.94, p=0.06) but at the voxel level (87 mm³) correlation was only moderate during exercise (r=0.73±0.08 vs. r=0.74±0.10, p=0.92) and weak at rest (r=0.28±0.13 vs. r=0.33±0.21, p=0.58). Exercise decreased both perfusion and FFA uptake heterogeneity within the muscles (p<0.001) similarly in both groups. In conclusion, long-term history of moderately increased physical activity tends to enhance muscle oxidative metabolism, but it does not have any significant influence on the FFA uptake or perfusion rates or their heterogeneity in skeletal muscle. Submaximal knee-extension exercise decreases heterogeneity of muscle FFA uptake and perfusion, and improves matching between local muscle perfusion and FFA uptake. Thus, it seems that the genetic influence is more important to determine the heterogeneity of perfusion and FFA uptake in skeletal muscle than exercise training.