Background <p>The autonomic synucleinopathy multiple system atrophy (MSA) can be difficult to distinguish clinically from Parkinson disease with orthostatic hypotension (PD+OH). ¹⁸F-Dopamine positron emission tomography separates these conditions based on cardiac noradrenergic deficiency in PD+OH and not in MSA but is available only at the NIH Clinical Center. 3,4-Dihydroxyphenylglycol (DHPG) is the main neuronal metabolite of norepinephrine. This retrospective observational study examined whether DHPG levels in cerebrospinal fluid (CSF) or plasma differentiate MSA from PD+OH.</p> Methods <p>We reviewed CSF and plasma neurochemical data from all patients referred for evaluation at the NIH Clinical Center between 1995 and 2024 for chronic autonomic failure or parkinsonism. A concurrently studied comparison group included healthy volunteers or patients with orthostatic intolerance.</p> Results <p>CSF DHPG was decreased in MSA (<i>N</i> = 67, <i>p</i> &lt; 0.0001) compared to the controls but also tended to be decreased in PD+OH (<i>N</i> = 31, <i>p</i> = 0.0776). Antecubital venous plasma DHPG was decreased in PD+OH (<i>N</i> = 47, <i>p</i> = 0.0064) but not in MSA. CSF/plasma concentration ratios of DHPG were lower in MSA than in PD+OH (<i>p</i> = 0.0005). Cardiac arteriovenous increments in plasma DHPG and cardiac norepinephrine spillovers were strikingly decreased in PD+OH (<i>N</i> = 6) and were lower than in MSA (<i>N</i> = 20, <i>p</i> &lt; 0.0001 each). Combining cardiac arteriovenous increments in plasma DHPG with norepinephrine spillovers completely separated PD+OH from MSA.</p> Conclusions <p>CSF/plasma ratios of DHPG, cardiac arteriovenous increments in plasma DHPG, and cardiac norepinephrine spillovers separate MSA from PD+OH. On the basis of our results we propose that biomarker combinations involving DHPG in biofluids may enable a clinical laboratory distinction of MSA from PD+OH.</p>