In vivo studies selectively assessing preganglionic and central autonomic nervous system activity in patients with autonomic failure have so far been limited to testing pituitary function. In animal experiments carbon dioxide (CO2) selectively stimulates central sympathetic nuclei in the ventrolateral medulla and preganglionic sympathetic neurons in the cervical trunk. This central stimulation seems to overrule less pronounced peripheral vasodilatatory effects. This study addressed the question of whether hypercapnea is a suitable challenge procedure to test preganglionic and central autonomic activity in healthy subjects and in patients with autonomic failure of preganglionic and central origin. Seven patients with multiple system atrophy (MSA) and 30 age-matched healthy volunteers underwent a protocol including a Valsalva manoeuvre (VM) under normo- and hypercapnic conditions and exposure to hypercapnea under supine resting conditions. Blood pressure (BP), heart rate (HR) and end-tidal CO2 partial pressure were measured continuously and non-invasively. In normal controls hypercapnea induced significantly higher BP values in phases II, IIe, III and IV of the VM compared to the normocapnic VM and a significant increase in BP during steady-state supine exposure compared to normocapnic baseline. HR increased significantly only after 40 s of steady-state hypercapnea during the latter challenge. In patients with MSA and autonomic failure, in whom a predominantly preganglionic lesion of the autonomic nervous system is established, no significant effects of hypercapnea on the cardiovascular parameters were found. Although this non-invasive challenge procedure cannot differentiate between pre- and postganglionic autonomic failure, exposure to hypercapnea enables the investigation of efferent autonomic activity to vasoconstrictors generated from autonomic centres in the brainstem and cervical trunk.