OBJECTIVES: Transcutaneous auricular vagus nerve stimulation (taVNS) modulates central and peripheral neurophysiology. Specifically, taVNS increases heart rate variability (HRV) indicating a shift in autonomic function towards parasympathetic predominance. However, knowledge on the influence of stimulation parameters and targets is scarce. We hypothesized that the location and charge per phase of taVNS influences HRV. MATERIALS AND METHODS: In thirteen healthy subjects, six different stimulation targets were investigated, i.e., cymba conchae, cavum conchae, outer tragus, inner tragus, crus helicis, and fossa triangularis. At each target, 24 parameter combinations were studied: Eight different electrical charges per phase were evaluated by investigating three pulse durations and eight charge-balanced current intensities, i.e., 100 mus (0.250-2 mA in steps of 0.250 mA), 260 mus (0.096-0.769 mA in steps of 0.096 mA), and 500 mus (0.050-0.400 mA in steps of 0.050 mA). In a parallel group design, left and right taVNS were compared to each other. 30 bursts at each parameter combination were applied with a periodicity of 1 Hz. Each burst consisted of five pulses applied at 25 Hz. RESULTS: HRV increased in a charge-dependent way with significant differences between the right and left ear. The targets with the strongest effects were the cymba conchae and fossa triangularis, and to a lesser extent the inner tragus. CONCLUSIONS: HRV is suitable to define taVNS parameters and targets for research and therapeutic purposes. Bursts of taVNS with a pulse duration of 100 mus and a current intensity of 2 mA are comfortable for the participants and effective in increasing HRV when applied at specific auricular locations. These findings need to be replicated in larger cohorts, and with longer stimulation and off-periods between conditions. Since results may differ in conditions with an impaired autonomic tone, future studies should also consider aged and patient populations.