This single-center randomized controlled trial aims to investigate the efficacy of transcutaneous auricular vagus nerve stimulation in alleviating nausea in healthy adults.

The primary aim of this study is to assess the efficacy of taVNS in reducing nausea in healthy adults subjected to nausea induction through intragastric lipid infusion, compared to sham stimulation, as measured by 0-100 Visual Analogue Scale (VAS) scores.

The secondary objectives include evaluating the potential of taVNS to alleviate other gastrointestinal symptoms, such as abdominal pain, bloating, and fullness, as well as exploring its effects on the desire to eat, all measured using 0-100 VAS scores. Additionally, changes in autonomic parameters, plasma levels of ghrelin and motilin, and salivary cortisol will be evaluated. The relationship between the nausea response and affective symptoms, as well as personality traits, will also be explored.

Participants will be randomly assigned to either the taVNS or the sham stimulation group, with the intervention administered for 30 minutes immediately following nausea induction through intragastric lipid infusion.

Visit 1 Interested study participants will receive a verbal explanation of the study over the phone and will be sent a comprehensive electronic information letter. At least 7 days later, they will be invited to attend an inclusion visit at Maastricht University. During this visit, participants will undergo eligibility screening and receive detailed instructions regarding the study procedures. Eligible participants will be asked to provide written informed consent before proceeding. Subsequently, eligible participants will be asked to complete several (digital) questionnaires, including a baseline characteristics questionnaire, the Generalized Anxiety Disorder 7-Item Scale (GAD-7), the Patient Health Questionnaire-9 (PHQ-9), and the Big Five Inventory (BFI). All information obtained during the screening visit, including participant data and study-related details, will be securely recorded using Castor, an electronic data capture system. After this screening visit participants will be randomized in a 1:1 parallel design by an investigator to receive either taVNS or sham stimulation on the second test day, with appropriate blinding procedures in place.

Visit 2 A second visit, which serves as the test day, will be scheduled. During this visit, participants will receive either taVNS or sham stimulation, administered immediately after nausea induction, based on their randomization.

Participants will arrive at the NUTRIM CRU at Maastricht University after an overnight fast. Compliance with test day regulations will be checked (e.g. fasting, no alcohol). Once compliance is confirmed, the coordinating investigator will properly set up the tVNS device to ensure it can be used immediately after nausea induction. This setup includes adjusting the stimulation current of the tVNS device according to each participant's sensitivity, ensuring it remains below the sensitivity threshold, with current intensities ranging from 0.25 to 10 mA. This is important because active stimulation will be programmed to deliver only subthresholdstimuli. Next, an intravenous cannula will be placed for blood collection. Thereafter, the investigator (a medical doctor) will manually place a nasogastric tube to facilitate the intragastric infusion of a 50% fat emulsion in water. This method of infusion is selected to replicate the conditions under which dietary fat is ingested as part of a meal. The infusion will be administered at a rate of 5 ml/min, consistent with previous research in which nausea was induced through similar methodologies (1). Participants will be closely monitored throughout the infusion for the onset of nausea, with assessments every 5 minutes using a 0-100 Visual Analogue Scale (VAS) to quantify the nausea severity. Participants will receive clear instructions on how to rate their nausea and indicate its intensity. If a predetermined VAS score of 50/100 is reached, or should the participant start retching, the lipid infusion will be promptly halted to prevent excessive discomfort. Participants will be informed in advance about the 50/100 VAS threshold and provided with guidance on how to interpret and apply this rating. Following the cessation of the infusion, the nasogastric tube will be removed for patient comfort, after which either taVNS or sham stimulation will be administered for 30 minutes.

A duration of 30 minutes was selected based on the mechanism of action of taVNS, which is assumed to act via the activation of the nucleus of the solitary tract (NTS) in the brainstem. Furthermore, this choice is also consistent with other studies on taVNS conducted within our department (NL87188.068.24/METC 24-029). The NTS serves as the primary relay station for vagal input and plays a critical role in modulating autonomic functions. In a previous study by Frangos et al. (24), NTS activity gradually decreased during stimulation but peaked shortly after the cessation of a 7-min stimulation of the cymba conchae. Furthermore, other brain regions exhibited a gradual increase in activity during stimulation, reaching their peak during the post-stimulation phase, with this heightened activity persisting for up to 11 minutes. Hence, a stimulation period of 30 minutes is expected to provide an optimal timeframe for taVNS to exert its maximum effect and to counterbalance the effects of nausea.

Both the taVNS treatment and sham stimulation will be administered using a tVNS device that provides stimulation to the cymba conchae of the left ear. The left side is chosen due to the innervation pattern of the left vagus nerve, which predominantly innervates the stomach and the pyloric area (25, 26). This choice aligns with our ultimate clinical interest in conditions such as gastroparesis and functional dyspepsia. Additionally, it ensures comparability with other studies conducted within our department, including the "tVNS and GI motility" study (NL86446.068.24).

The stimulation parameters comprise a biphasic rectangular pulse wave with an impulse frequency of 25Hz, impulse duration of 30 seconds, impulse pause of 30 seconds, and pulse width of 450ms. These parameters have been identified as the most optimal treatment frequencies for taVNS based on systematic reviews (27, 28). Active stimulation will be programmed to deliver only subthreshold stimuli. The device will blink when stimulation is delivered. In the sham (control) group, the electrode will be non-conducting, but the device will still blink to maintain blinding (28). The electrodes used in the tVNS and sham groups differ slightly. Consequently, the investigator responsible for programming the devices will be aware of the treatment allocation, while participants will remain blinded. To ensure double blinding, the randomization code will be altered prior to data analysis, ensuring that the investigator conducting the analysis will be blinded to the treatment allocation. The randomization code will be kept confidential until after the modified intention-to-treat analysis has been reported.

During the stimulation phase, nausea severity will be assessed using a 0-100 VAS score, where 0 indicates "no nausea" and 100 represents the "worst nausea imaginable". VAS scores will be recorded every 5 minutes, continuing for 30 minutes post-stimulation to evaluate the efficacy of taVNS in alleviating nausea. Additionally, other gastrointestinal symptoms and the desire to eat will be measured at predetermined time points using VAS scores.

Blood samples will be taken at four time points to assess changes in plasma ghrelin and motilin levels, selected for their presumed roles in the physiological response to nausea (3). Ghrelin is known to stimulate appetite, and since nausea is often accompanied by a decrease in appetite, it is likely that nausea leads to reduced ghrelin levels. Measuring ghrelin in response to taVNS will allow us to investigate whether this intervention influences the hormonal response and potentially uncover the mechanism by which taVNS alleviates nausea. Motilin is a gastrointestinal hormone primarily involved in regulating gastric motility and stimulating the migrating motor complex (MMC) during the fasting state (11). Although not previously investigated directly, it may play a role in the nausea response through its effects on gastric motility (29, 30). Levels of motilin, along with gastrointestinal motility, are likely to be altered in response to intragastric lipid infusion and the subsequent onset of nausea and a decrease in gastric emptying. TaVNS may modulate motilin levels, as it is expected to reduce nausea and influence the MMC (30). It is hypothesized that this reduction in nausea may correspond with an increase in motilin levels, facilitating the restoration of normal gastric motility (29). By measuring both hormones, we aim to gain deeper insights into the physiological effects of taVNS on the nausea response.

Saliva samples will be collected at the same four time points as the blood samples to assess changes in cortisol levels. Cortisol plays a crucial role in regulating the stress response. Nausea is considered a stressor, activating both the autonomic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis (60). As a result, cortisol could serve as an important marker for the degree of physiological dysregulation associated with nausea. By measuring cortisol, it can objectively be assessed to what extent nausea triggers a stress response and whether taVNS has a regulatory effect on this process. This provides valuable additional information regarding hormonal and autonomic changes, contributing to a deeper understanding of the potential physiological effects of taVNS.

Autonomic parameters, including heart rate variability and skin conductance, indicators of both parasympathetic and sympathetic activity, will be recorded continuously from the start of nausea induction until 30 minutes after either taVNS or sham stimulation. These measurements will be captured using a Shimmer3 GSR sensor and a Fitbit smartwatch.

After completing all measurements, participants will be asked to fill out the reimbursement form. Phone numbers of the investigators and the emergency department will be provided for any issues that arise within 24 hours after the test day. The investigators will ensure participants are in a stable condition before leaving Maastricht University.

Inclusion Criteria:

• Healthy participants (defined as those without a pre-existing medical comorbidity).
• Aged between 18-65 years.
• Ability to understand and speak the Dutch language.
• BMI between 18 and 25 kg/m2

Exclusion Criteria:

• Medical history of chronic or severe diseases affecting the cardiovascular, respiratory, urogenital, gastrointestinal/hepatic, haematologic/immunologic, HEENT (head, ears, eyes, nose, throat), dermatological/connective tissue, musculoskeletal, metabolic/nutritional, endocrine, neurological/psychiatric systems.
• A history of major abdominal surgery.
• Gastrointestinal complaints.
• Any use of medication, especially those affecting gastric motility and nausea, apart from oral contraceptives.
• Current or lifetime psychopathology (including PHQ-9 and GAD-7 scores > 10)
• Substance abuse, including excessive alcohol consumption (>20 alcoholic consumptions per week) and the use of recreational drugs.
• Smoking.
• Pregnancy, lactation, or intention to become pregnant during the study period
• Use of devices (e.g., cochlear implants) or other conditions (e.g. wounds, permanent ear-piercing) complicating the use of the tVNS device.
• Administration of investigational drugs or participation in any scientific intervention study that might interfere with this study (to be determined by the principal investigator) within 180 days preceding the commencement of the study.
• Students and employees of Maastricht University are not precluded from participation, unless they have a direct personal, professional or hierarchical position with regards to any of the study team members or their department.