Examination of peripheral nerves with high-resolution ultrasound (HRUS) now routinely complements electrophysiological studies in various diseases. Prerequisite for nerve HRUS are well-defined reference values. Notably, reference values of the vagus nerve (VN) are varying (Cartwright, 2008; López-Hernández, 2014) and, overall, poorly studied. Aim of this study was to assess reference values for the VN as well as to examine inter-rater and intra-rater reliability and inter-ultrasound system agreement.Both VNs of 60 healthy subjects (37 female, mean age 49.6±19.5years, range 22–76) were examined with two high-resolution ultrasound systems (Toshiba Aplio 500, 18MHz probe and Esaote MyLab Five, 15MHz probe) by two sonographers experienced in peripheral nerve ultrasound. Cross-sectional area (CSA) of each VN was assessed at 2 points: at the level of the carotid sinus (proximal measuring point) and at the level of the thyroid gland (distal measuring point). Three ultrasound images were recorded at each measuring point. Subsequently, the best scan was used for further offline measurements.At both measuring levels the mean CSA of the VN was significantly larger on the right (proximal: 2.7±0.6mm2/2.7±0.5mm2 [examiner 1/ examiner 2], distal: 2.6±0.6mm2/2.6±0.5mm2) than on the left side (proximal: 2.1±0.5mm2/2.2±0.5mm2, distal 1.9±0.4mm2/2.0±0.4mm2; student”s paired t-test, each p<0.01). Overall, there were a good inter-rater agreement (intraclass correlation coefficient [ICC] 0.72, 95% confidence interval [CI] 0.65–0.77), a good intra-rater agreement (ICC 0.81, 95% CI 0.74–0.86) and a good agreement between both ultrasound systems (ICC 0.82, 95% CI 0.75–0.87). Notably, agreement was consistently superior at the level of the thyroid gland.We recommend examining the VN at level of the thyroid gland where it can reliably be visualized. However, since most ultrasound systems calculate the CSA only in full mm2 which would not be accurate enough for the small VN, measurements should be done off-line with appropriate software. Despite those general considerations, the VN is the first nerve with asymmetric reference values. These side-specific findings might probably be due to the unpaired vagal innervation of the thoracic and abdominal organs. Moreover, due to a good inter- and intra-rater as well as inter-ultrasound system agreement, our side-specific reference values will be useful for future trials that examine the VN in the context of neurodegenerative or autoimmune diseases.