In the field of canine rehabilitation, knowledge of muscle function in the therapeutic exercises prescribed is needed by physical therapists and veterinary surgeons. To gain insight into the function of longissimus dorsi (LD) and gluteus medius (GM) muscles in dogs, five Greyhounds performing leash walking and trotting on the ground flat, up (+7%), and downhill (−7%) were studied by surface electromyography, and the mean and maximum activity was compared. For the same incline, the surface electromyography (sEMG) of LD was higher (p < 0.05) at the trot than at the walk. In LD muscle, trotting uphill showed significantly higher maximum activity than any other exercise. A change of +7% incline or −7% decline affected (increased or decreased, respectively) the mean sEMG of the LD and GM muscles of dogs walking or trotting on the ground. When combined, the influence of gait and incline on electromyographic activity was analyzed, and walking at certain inclines showed no difference with trotting at certain inclines. Walking and trotting up and downhill added separate therapeutic value to flat motion. The results of the present study might contribute to a better understanding of the function of LD and GM muscles in dogs, this being especially useful for the field of canine rehabilitation.