An acute bout of aerobic exercise induces neuroplasticity in the motor cortex. Moreover, paired associative stimulation (PAS) is known to induce neuroplasticity in M1. However, the possible influence of the type of exercise on the neuroplastic changes remains unknown. The present study investigated the effects of two different modes of muscle contraction produced during locomotor exercise on changes in corticospinal (CS) excitability. Subjects performed two 30-min treadmill exercises at an intensity corresponding to 60% of their maximal heart rate with either a +10% (uphill) or −10% (downhill) slope. These exercises were followed or not by paired associative stimulation method (PAS25) which consisted of 200 paired stimuli (0.25 Hz, 15 min) of median nerve electrical stimulation followed by transcranial magnetic stimulation of the hand M1 area (ISI 25 ms). Motor evoked potentials (MEP), assessed through abductor pollicis brevis (APB) activity were obtained before exercise, at 5 min, 15 min and 30 min after exercise. A significant (P < 0.05) increase of the MEP amplitude was observed 30 min after both exercises but was not different between the two modes of locomotion. On the contrary, MEP amplitude with PAS25 increased only 30 min after downhill exercise. We conclude that sub-maximal treadmill exercise increases CS excitability within a period of 30 min. However, the predominant mode of muscle contraction during uphill versus downhill locomotion does not influence CS excitability when assessed using a non-exercised muscle. However, results from PAS25 suggest that specific neuroplastic changes occur likely due to homeostatic mechanisms induced by exercise plus a PAS protocol.