BACKGROUND AND OBJECTIVES: Diagnosing dementia with Lewy bodies (DLBs) is challenging because of symptom overlap with other neurodegenerative diseases. Although dysautonomia is a recognized supportive diagnostic criterion, its prevalence and extent remain underexplored. We aimed to evaluate autonomic dysfunction in patients with DLB using a comprehensive battery of autonomic function tests (AFTs) and to investigate whether this dysfunction differs between patients with and without nigrostriatal denervation. METHODS: This prospective cohort study was performed at a Memory Clinic in Rome, Italy. Patients meeting diagnostic criteria for possible DLB were enrolled and underwent AFTs including head-up tilt test (HUTT), Valsalva maneuver, deep breathing, cold face, hand grip (HG), and electrochemical skin conductance. Dopamine transporter SPECT (DaT-SPECT) was performed to assess nigrostriatal transmission. We compared results from AFTs in (1) patients with DLB vs healthy controls (HCs) and (2) patients with DLB with pathologic vs normal DaT-SPECT. RESULTS: Twenty-two patients with DLB (median age: 72.00 [10.00] years, %female: 21.75) and 20 HCs (median age: 69.00 [5.25] years, %female: 40) were enrolled. Only 1 patient (4.5%) showed neurogenic orthostatic hypotension (nOH) at HUTT. However, patients with DLB showed cardiovascular adrenergic dysfunction, represented by lower Valsalva overshoot ( DISCUSSION: Despite the absence of overt nOH, patients with DLB show covert dysautonomia encompassing adrenergic, parasympathetic, and sudomotor dysfunction, highlighting the importance of standardized autonomic evaluation. Patients with normal DaT-SPECT exhibited greater peripheral autonomic impairment, reflected by lower HG responses, suggesting diverse α-synuclein pathology trajectories within DLB. Further research is needed to explore autonomic nervous system dysfunctions across different DLB subtypes and stages.