Less heterogeneous skeletal muscle perfusion has recently been reported in endurance-trained compared to untrained men at macrovascular level. The causes of this difference in perfusion heterogeneity are unknown as is whether the same difference is observed in microvasculature. We hypothesised that the difference could be caused by changes in muscle vascular branching pattern. Perfusion was measured in resting and exercising muscle in 14 endurance-trained and seven untrained men using [¹⁵O]water and positron emission tomography. Fractal dimension (D) of perfusion distribution was calculated as a measure of fractal characteristics of muscle vascular branching pattern. Perfusion heterogeneity in microvascular units (1 mm³ samples) was estimated using the measured heterogeneity in voxels of PET images (relative dispersion, RD = S.D./mean) and corresponding D values. D was similar between the groups (exercising muscle 1.11 ± 0.07 and 1.14 ± 0.06, resting muscle 1.12 ± 0.06 and 1.14 ± 0.03, trained and untrained, respectively). Trained men had lower perfusion (151 ± 44 vs. 218 ± 87 ml min^-1 kg^-1, P < 0.05) and macrovascular perfusion heterogeneity (relative dispersion 21 ± 5 vs. 25 ± 5 %, P < 0.05) in exercising muscle than untrained men. Furthermore, estimated perfusion heterogeneity in microvascular units in exercising muscle was also lower in trained men (33 ± 7 vs.48 ± 19 %, P < 0.05). These results show that fractal vascular branching pattern is similar in endurance-trained and untrained men but perfusion is less heterogeneous at both the macro- and the microvascular level in endurance-trained men. Thus, changes in fractal branching pattern do not explain the differences in perfusion heterogeneity between endurance-trained and untrained men.