Examining Transcutaneous Vagal Nerve Stimulation as a Facilitator of Social Bonding
Conditions: Vagus Nerve Stimulation
Sponsor: University of California, San Francisco

 
 
 
 
 
 
 
Clinical Trials Study Protocol 
Examining Transcutaneous Vagal Nerve Stimulation as a Facilitator of Social Bonding 
(NCT05899413) 
 
 
 
 
Version 1 
March 1, 2025 
 
 

 
This study was approved by the Institutional Review Board at the University of 
California, San Francisco (#23-39189). We conducted a power analysis prior to the data 
collection to determine the power for the Level-2 direct effect (stimulation condition) based on 
1,000 simulations using the package simr in R (R Core Team). Given the nature of our dyad and 
the outcomes (i.e., questions about the same interaction), we assumed a large intraclass 
coefficient (0.5). Following Arend and Schäfer’s (2019) recommendations, the standardized 
Level-1 direct effect was set to small (γstd. = .10) in our power estimation for the Level-2 effect. 
Our analysis suggested that 70 dyads will provide > 82% power (α set to .05) to detect a 
medium-to-large standardized Level-2 direct effect (γstd. = .40).  
Data collection for the study took place between February and June 2024. Participants 
were recruited in the San Francisco Bay Area through various community and university 
newsletters and listservs, flyers around the city, Reddit, and word of mouth. We tried to reach 
diverse populations by promoting the study through various community centers (e.g., SF LGBT 
Center) and universities in the city (e.g., San Francisco State University, UC Berkeley). 
Eligibility was confirmed using a screening questionnaire. Participants were eligible for 
the study if both they and their partner were between 18 and 35 years old, fluent in English, had 
no major chronic diseases (e.g., cardiovascular disease) or untreated psychiatric diagnoses, were 
not taking any confounding medications (e.g., antihypertensive medications), and did not have 
unremovable piercings on the tragus. We restricted the age range to control for age-related 
differences in physiological responses. As HRV tends to decrease with age, we attempted to 
recruit a more homogenous group on one of our primary outcome variables (HRV level and 
reactivity), so we capped the age range to mid-adulthood. Participants were also required to have 

been in a relationship with their partner for at least one year and be able to complete the in-
person laboratory visit together.  
Tasks and Measures 
Pre-visit survey. Prior to the lab visit, participants completed pre-visit surveys that 
collected information on basic sociodemographic characteristics, relationship characteristics, and 
physical attributes, including height and weight. Self-reported height and weight were used to 
compute Body Mass Index (BMI; weight (lb) ÷ height2 (inches) * 703), which we used as a 
covariate. Participants also responded to a 5-item measure of relationship satisfaction (e.g., “I 
feel satisfied with our relationship”; α = .81) and a 7-item measure of commitment (e.g., “I want 
our relationship to last for a very long time”; α = .79) from the Investment Model Scale (Rusbult 
et al., 1988), which we used to test differences between participants in the experimental and 
control conditions. Additionally, they rated 24 relationship issues (e.g., communication, family 
and in-laws; adapted from Geiss & O’Leary, 1981) on a scale ranging from 0 (issues that rarely 
if ever raise conflict or disagreement) to 10 (issues that raise frequent or intense conflict or 
disagreement). Participants’ responses to this scale were used in the conflict interaction described 
below. 
Gratitude interaction. The procedures were based on Algoe and colleagues (2013). 
Couples were told to think about a positive thing their partner did for them recently for which 
they felt grateful. They were given three minutes to prepare, then took turns to express their 
gratitude for three minutes each. While one partner was speaking, the listening partner was told 
to listen with minimal verbal reactions.  
Conflict interaction. The procedures were based on Levenson and Gottman (1983) and 
previous studies that examined physiological reactivity during conflicts (e.g., Godfrey & 
Babcock, 2020; Raby et al., 2015). Couples were told that they would discuss a conflict topic 

relevant to their relationship and try to resolve it. The interaction would involve the following 
steps: 1) couples selecting a topic to discuss together, 2) reflecting on the topic independently for 
three minutes, 3) each partner taking turns expressing their thoughts and feelings about the topic 
for one minute (the speaking order was predetermined based on the alphabetical order of their 
first names), and 4) couples engaging in free discussion for six minutes, with the goal of 
resolving the issue.  
 
After receiving the instructions, couples were given a list of three topics to choose from. 
Unknown to the couples, this list was generated using participants’ responses regarding the 
frequent topics of arguments that they provided in the pre-visit survey. The three topics were 
chosen based on the highest average ratings of the two partners. If no topics averaged higher than 
3, the list included an option of “any topic that recently triggered a conflict or disagreement in 
the relationship.” 
 
Following the interaction, participants completed a short questionnaire that included 
measures of positive affect (“During the conversation, overall, I felt…”; 1: not at all positive, 7: 
extremely positive) and negative affect (“During the conversation, overall, I felt…”; 1: not at all 
negative, 7: extremely negative). 
tVNS device and stimulation. We used a research edition of an auricular vagus nerve 
stimulator (tVNS RE, Vagus.net™, UK) that sends current-controlled stimulation in symmetric 
biphasic waveforms. The tVNS RE conforms to EU directive 93/42/EEC medical device 
standards and ISO/IEC 17050-1. This device consists of a handheld stimulator and an electrode 
clip. Following previous research (Kraus et al., 2007), couples in the sham stimulation condition 
had the electrode clip placed on the center of their left earlobe (free of cutaneous vagal 
innervation; Peuker & Filler, 2002) while those in the tVNS condition had the electrodes placed 

on their tragus. While cymba concha has also been used for tVNS, there is no evidence that 
psychophysiological effects of tVNS depend on the stimulation site (Borges et al., 2021). In fact, 
tVNS effects on executive functioning may be larger when stimulation is applied at the tragus 
rather than cymba concha (Ridgewell et al., 2021). We stimulated the left ear, which has been a 
common practice as the efferent vagal fibers to the heart are located on the right side (Kim et al., 
2022) but note that the side effects are considered negligible and studies have also used right-
sided stimulation (see Kaduk et al., 2023).  
The experimenter first cleaned the stimulation loci with alcohol cotton swabs to reduce 
skin resistance. To identify the lowest stimulation intensity that participants could perceive, we 
adopted the staircase procedures used in previous research (e.g., Schuerman et al., 2021). 
Specifically, the experimenter placed the electrode clip on the participant’s left ear (tragus or 
earlobe) and increased the stimulation levels by 0.1mA, starting from zero, until the participant 
reported feeling a tingling sensation. Experimenters confirmed the threshold twice, and the 
stimulation level was set 0.2mA below each participant’s perceptual threshold (Schuerman et al., 
2021) to allow couples to converse as naturally as possible and to attempt to keep participants 
unaware of their condition assignment. We delivered stimulation at the individually calibrated 
intensity continuously during the interaction, and the default device settings we adopted were as 
follows: frequency = 30Hz, phase duration = 250μs, interphase interval = 50μs, and max voltage 
= 65V.  
Physiological data acquisition. Participants’ physiological responses were continuously 
recorded during the baseline and all interactions. We collected electrocardiography (ECG) and 
impedance cardiography, which were integrated using an MP150 system (Biopac Systems Inc., 
Goleta, CA). Spot sensors were placed in a modified lead Ⅱ configuration on the torso (near the 

right clavicle and below the left rib cage) to measure ECG (Mendes, 2009). Data were acquired 
using Acqknowledge software at a sampling rate of 1000Hz (Biopac Systems Inc, Goleta, CA, 
USA). RSA was edited and scored in 30-second intervals using the HRV module from Mindware 
Technologies (Gahanna, OH), which identified the R spikes of each heartbeat in the ECG. 
Although 60-second epochs are generally recommended, 30-second epochs have been reliably 
used in previous dyadic studies (Caldwell et al., 2018; Oshri et al., 2023; Qaiser et al., 2023) and 
may sufficiently capture high-frequency activity especially when the heart rate is elevated (i.e., 
shorter heart period; Quigley et al., 2024), as in conflict settings. After data collection, trained 
research assistants blind to the condition visually inspected all data for artifacts and edited them 
as needed (e.g., removal of misplaced R-peaks).  
Post-study survey. At the end of the study, before debriefing and being informed of the 
condition they were assigned to, participants answered a few questions about their experience of 
the stimulation and the tVNS device. Specifically, we asked about awareness of the stimulation 
(“How much, if at all, could you feel the tingling sensation during the conversations?”), general 
discomfort (“How uncomfortable was wearing the tVNS device [i.e., the clip you had on your 
ear] throughout the study?”), perceived influence of discomfort on interactions (“How much, 
if at all, did the discomfort [from wearing the clip] affect your conversations?”), and perceived 
influence of tVNS on interactions (“How much, if at all, do you think the device changed the 
way you felt or interacted with your partner during the conversations?”). All items were rated on 
a 5-point scale. 
Study Procedure 
Interested participants first completed a screening questionnaire to confirm eligibility. 
Eligible participants were then contacted via email to schedule a lab visit. Two days prior to the 

scheduled visit, participating couples received a link to a consent form and a questionnaire 
assessing sociodemographic information and relationship characteristics. Couples were randomly 
assigned to either tVNS or sham stimulation condition when they both completed the pre-visit 
survey. Each couple had an equal chance of being in either group.  
Upon arrival at the lab, partners were escorted to separate rooms where they each 
received a full overview of the study and underwent threshold testing for stimulation. Then 
partners reunited in the same room, were connected to physiological sensors, and spent five 
minutes, resting in a comfortable seated position (baseline). As shown in Figure 1, they were 
seated at an angle, partially facing each other, with their bodies slightly tilted toward the front 
where a fixed wall-mounted camera was positioned. Participants were recorded during gratitude 
and conflict interactions. Full study procedure, including the timing of the stimulation is 
illustrated in Figure 2. Following a cool-down period, participants completed a brief 
questionnaire about their experience with the stimulation and were debriefed. They also signed 
an additional consent form for the use of their recorded videos.  
 
 
Figure 1. Example Positioning of the Couple During the Study 

 
 
 
Figure 2. Overview of Study Procedure 
Notes. The time next to the lightning icon indicates the duration of the (active or sham) 
stimulation. The time at the bottom represents the duration of each study segment described 
within the rectangles. The survey icon indicates when participants complete short questionnaires.  
 
Statistical Analysis Plan 
All analyses will be conducted in R (R Core Team, 2022). Multilevel model analyses will 
be conducted using the packages nlme and lmer. All tests will be two-tailed, with a significance 
threshold of α = .05.  
Randomization check. We will first examine whether there are condition differences in 
personal and relational characteristics at baseline, including age, relationship duration, global 
relationship satisfaction, and commitment. To account for the interdependence of the dyadic data, 
we will fit a series of multilevel models with a random intercept included for participants nested 
within couples and condition included as a fixed effect. Restricted Maximum Likelihood will be 
used to fit the model parameters. For relationship duration, which is a couple-level variable, 
Welch’s t-test will be conducted.  

Study artifact check. We will examine if there are condition differences in participants’ 
experiences related to the stimulation and the tVNS device reported at the end of the study, 
including awareness of the stimulation, general discomfort, perceived influence of discomfort on 
interactions, and perceived influence of tVNS on interactions. We will run a parallel set of 
multilevel models as described above. 
tVNS effects on emotional experience of the conflict. We will examine if there are 
condition differences in how couples self-report their experience of the conflict interaction, 
specifically in terms of positive and negative affect during the interaction. We will run a parallel 
set of multilevel models as described above. 
tVNS effects on physiological reactivity. As in previous research, we will calculate 
reactivity scores by subtracting the raw RSA score during the last 30 seconds of baseline from 
every segment of the interaction. We will fit a multilevel model, first estimating the effect of 
condition (coded as -0.5 for sham and 0.5 for tVNS) on RSA reactivity with no covariates. As in 
previously described multilevel models, we will include a random intercept for participants 
nested within couples, and as recommended for physiological data, with autoregressive 
covariance structure of the residuals. In our follow-up models, we will control for sex 
(male/female/other), age, BMI, and relationship duration, which can influence physiological 
reactivity to conflicts. Then, to rule out the possibility that any condition effects are reducible to 
differences in the experience of the stimulation, the next model will additionally control for 
stimulation intensity and discomfort reported at the end of the study. We will repeat these 
analyses using IBI in place of RSA. 
 
 

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