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.