We have recently shown that neurogenic vasodilatation in non-contracting and contracting vastus lateralis (VL) muscles is induced by descending signal from higher brain centres (termed central command) at the start of voluntary one-legged cycling and during motor imagery of the exercise (Ishii et al. 2012). The centrally-induced vasodilatation may be mediated by a sympathetic cholinergic mechanism as our laboratory reported previously using voluntary static exercise in conscious cats (Komine et al. 2008). On the other hand, as another possibility, central command may activate sympathetic adrenergic nerve, which in turn causes β-adrenergic vasodilatation in skeletal muscle. We examined which of the two vasodilator mechanisms contributes to the centrally-induced vasodilatation in both non-contracting and contracting VL muscles at the start of voluntary one-legged cycling with the right leg and during motor imagery of the cycling. The blood flow responses in the VL muscles during the two interventions were compared before and after intravenous injection of atropine sulfate (10 μg/kg) or propranolol (0.1 mg/kg). The relative concentrations of oxygenated- and deoxygenated-hemoglobin (Oxy- and Deoxy-Hb) in the bilateral VL muscles were measured with near-infrared spectroscopy as an index of muscle tissue blood flow in nine subjects. The Oxy-Hb increased by 3.5 ± 1.2% (mean ± S.E.M.) in the non-contracting VL muscle and by 3.1 ± 2.0% in the contracting VL muscle at the start of the one-legged cycling, although the Deoxy-Hb was unchanged throughout the cycling. The results suggested the increased in tissue blood flow to both contracting and non-contracting VL muscles. Atropine abolished the initial increases in the Oxy-Hb of both non-contracting and contracting VL muscles. In contrast, propranolol did not affect the initial hyperaemia in both VL muscles but tended to reduce the Oxy-Hb responses during the later period of one-legged cycling, suggesting that the initial hyperaemia cannot be explained by β-adrenergic vasodilatation. Motor imagery of the right one-legged cycling caused increases in the Oxy-Hb of bilateral VL muscles (right VL: 2.4 ± 0.5%; left VL: 3.1 ± 0.6%) without changing the Deoxy-Hb. The centrally-induced increases in Oxy-Hb were abolished by atropine, but they were not affected by propranolol. Taken together, it is concluded that central command transmits cholinergic, but not β-adrenergic, vasodilator signals to both non-contracting and contracting skeletal muscle at the start of voluntary exercise and during motor imagery.