BACKGROUND: Both dopaminergic medication and subthalamic nucleus (STN) deep brain stimulation (DBS) can improve the amplitude and speed of gait in Parkinson disease (PD), but relatively little is known about their comparative effects on gait variability. Gait irregularity has been linked to the degeneration of cholinergic neurons in the pedunculopontine nucleus (PPN). OBJECTIVES: The STN and PPN have reciprocal connections, and we hypothesized that STN DBS might improve gait variability by modulating PPN function. Dopaminergic medication should not do this, and we therefore sought to compare the effects of medication and STN DBS on gait variability. MATERIALS AND METHODS: We studied 11 patients with STN DBS systems on and off with no alteration to their medication, and 15 patients with PD without DBS systems on and off medication. Participants walked for two minutes in each state, wearing six inertial measurement units. Variability has previously often been expressed in terms of SD or coefficient of variation over a testing session, but these measures conflate long-term variability (eg, gradual slowing, which is not necessarily pathological) with short-term variability (true irregularity). We used Poincaré analysis to separate the short- and long-term variability. RESULTS: DBS decreased short-term variability in lower limb gait parameters, whereas medication did not have this effect. In contrast, STN DBS had no effect on arm swing and trunk motion variability, whereas medication increased them, without obvious dyskinesia. CONCLUSIONS: Our results suggest that STN DBS acts through a nondopaminergic mechanism to reduce gait variability. We believe that the most likely explanation is the retrograde activation of cholinergic PPN projection neurons.