Purpose This study aimed to elucidate whether muscle activity (in terms of glucose uptake) between the legs can be considered symmetrical during walking. Furthermore, we aimed to determine whether the [18F]-fluorodeoxyglucose was distributed heterogeneously throughout each muscle, and if so, whether areas of high uptake would be clustered. Methods Ten healthy participants walked on a treadmill at self-selected comfortable walking speed for a total of 90 minutes, 60 minutes before and 30 minutes after intravenous injection of 50 MBq [18F]-fluorodeoxyglucose. Thereafter, a positron emission tomography/computed tomography scan of the lower limb was acquired. Three-dimensional muscle contours of 78 (= 39x2) muscles of the left and right lower limb were semi-automatically determined from magnetic resonance imaging scans. After non-rigid registration, those muscle contours were used to extract [18F]-fluorodeoxyglucose uptake from the positron emission tomography scans. Results Large asymmetries were observed in the lower leg muscles (e.g. median absolute asymmetry index of 42% in the gastrocnemius medialis) and in the gluteus minimus (30% asymmetry) and gluteus medius (15% asymmetry), whereas the uptake in the thighs was relatively symmetrical between the limbs (<6% asymmetry). These were not related to limb-dominance nor to inter-limb differences in muscle volume. The [18F]-fluorodeoxyglucose distribution was not distributed normally; most voxels had a relatively low standardized uptake value, and a minority of voxels had a relatively high standardized uptake value. The voxels with higher [18F]-fluorodeoxyglucose uptake were distributed heterogeneously; they were clustered in virtually all muscles. Conclusion The findings in this study challenge the common assumption of symmetry in muscle activity between the limbs in healthy subjects. The clustering of voxels with high uptake suggests that even in this prolonged repetitive task, different spatial regions of muscles contribute differently to walking than others.