ABSTRACT: A single bout of aerobic exercise modulates corticospinal excitability, intracortical circuits, and serum biochemical markers such as brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1). These effects have important implications for the use of exercise in neurorehabilitation. Here, we aimed to determine whether increases in cardiorespiratory fitness (CRF) induced by 18 sessions of high-intensity interval training (HIIT) over 6 weeks were accompanied by changes in corticospinal excitability, intracortical excitatory and inhibitory circuits, serum biochemical markers and working memory (WM) capacity in sedentary, healthy, young males. We assessed motor evoked potential (MEP) recruitment curves for the first dorsal interosseous (FDI) both at rest and during tonic contraction, intracortical facilitation (ICF), and short-interval intracortical inhibition (SICI) using transcranial magnetic stimulation (TMS). We also examined serum levels of BDNF, IGF-1, total and precursor (pro) cathepsin B (CTSB), as well as WM capacity. Compared to pretraining, CRF was increased and ICF reduced after the HIIT intervention, but there were no changes in corticospinal excitability, SICI, BDNF, IGF-1, total and pro-CTSB, and WM capacity. Further, greater CRF gains were associated with larger decreases in total and pro-CTSB and, only in Val/Val carriers, with larger increases in SICI. Our findings confirm that HIIT is efficacious in promoting CRF and show that corticospinal excitability, biochemical markers, and WM are unchanged after 18 HIIT bouts in sedentary males. Understanding how aerobic exercise modulates M1 excitability is important in order to be able to use exercise protocols as an intervention, especially in rehabilitation following brain injuries.