To explore the effects of qualitative or quantitative changes in walking on motor cortical excitability, a transcranial magnetic stimulation procedure was used to examine the alterations of motor-evoked potential (MEP) amplitude following walking. Eight healthy participants completed a series of two walking tasks on a treadmill at 2 km/h. The ratio of the left stance duration to the right stance duration was 1 : 2 in the asymmetrical walking task and 1 : 1 in the symmetrical walking task. In each task, walking for 10 min followed by MEP measurement for ∼4 min was repeated three times. MEP measurements were also performed before a walking task as a baseline and continued every 10 min for a further 30 min after the completion of the walking task. During slight voluntary contraction of the left tibialis anterior muscle, MEP measurements were conducted four times. Although a significant MEP depression was found after the asymmetrical walking task with increasing amount of walking, no significant decrease in MEP below baseline was observed after the symmetrical walking task throughout all measurement sessions. This MEP depression was the prominent response to the asymmetrical walking task compared with the symmetrical walking task. These findings indicate that the intentional control of walking pattern has both temporal and task-specific influences on excitability changes in the cerebral cortex, and suggest that motor cortical excitability may be altered by controlling the amount of central commands to the legs even during gait exercise.