OBJECTIVES: As a potential treatment for epilepsy, transcutaneous auricular vagus nerve stimulation (taVNS) has yielded inconsistent results. Combining transcranial magnetic stimulation with electromyography (TMS-EMG) and electroencephalography (TMS-EEG) can be used to investigate the effect of interventions on cortical excitability by evaluating changes in motor evoked potentials (MEPs) and TMS-evoked potentials (TEPs). The goal of this study is to objectively evaluate the effect of taVNS on cortical excitability with TMS-EMG and TMS-EEG. These findings are expected to provide insight in the mechanism of action and help identify more optimal stimulation paradigms.MATERIALS AND METHODS: In this prospective single-blind cross-over study, 15 healthy male subjects underwent active and sham taVNS for 60min, using a maximum tolerated stimulation current. Single and paired pulse TMS was delivered over the right-sided motor hotspot to evaluate MEPs and TEPs before and after the intervention. MEP statistical analysis was conducted with a two-way repeated measures ANOVA. TEPs were analyzed with a cluster-based permutation analysis. Linear regression analysis was implemented to investigate an association with stimulation current.RESULTS: MEP and TEP measurements were not affected by taVNS in this study. An association was found between taVNS stimulation current and MEP outcome measures indicating a decrease in cortical excitability in participants who tolerated higher taVNS currents. A subanalysis of participants (n=8) who tolerated a taVNS current ≥2.5mA showed a significant increase in the resting motor threshold, decrease in MEP amplitude and modulation of the P60 and P180 TEP components.CONCLUSIONS: taVNS did not affect cortical excitability measurements in the overall population in this study. However, taVNS has the potential to modulate specific markers of cortical excitability in participants who tolerate higher stimulation levels. These findings indicate the need for adequate stimulation protocols based on the recording of objective outcome parameters.