Non-invasive Auricular Fiber Vagus Nerve Stimulation (afVNS) for Treatment of Autism Spectrum Disorder: An Open-Label Trial
Phase: NA
Conditions: Autism Spectrum Disorder
Sponsor: Neuropix

 
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STUDY PROTOCOL 
Non-invasive Auricular Fiber Vagus Nerve Stimulation (afVNS) for Autism Spectrum 
Disorder: A Study Protocol for an Open-Label Trial Investigating Clinical and 
Physiological Effects 
 
Trial 
registration: 
This 
trial 
was 
registered 
in 
the 
clinical 
trials 
register 
http://www.clinicaltrials.gov (NCT06473623)    
 
Date of Document: 1st October 2025 
Protocol version: 1,10/ 2025 
 

 
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Objectives 
We hypothesize afVNS represents an adoptable, non-pharmacological adjunct therapy for 
home healthcare. We anticipate this intervention will support improvements in core autistic 
traits and commonly co-occurring health challenges.  
 
The objectives of this clinical trial are: 
1. To test the feasibility and adoption of afVNS for autistic individuals in the home setting 
2. To evaluate the short-term effects of afVNS on core autistic characteristics and 
associated conditions. 
3. To assess the impact of afVNS on ANS biomarkers in autism, as well as on related 
domains including sleep quality, verbal fluency and anxiety. 
 
These objectives correspond to specific aims regarding benefits and harms: 
• 
Harms: Objective 1 includes systematic assessment of safety and tolerability, with 
comprehensive monitoring and reporting of adverse events. 
• 
Benefits: Objectives 2 and 3 aim to collect preliminary data on potential clinical and 
physiological effects to inform future trial design.  
 
Design and Methods 
This is a single arm, open-label, exploratory study to be conducted at a single clinical site 
(Hong Kong). Following training and baseline assessments, afVNS feasibility and tolerance 
will be assessed over 14 days home healthcare application. The tolerability of afVNS 
application and completion of daily stimulation will be closely monitored, given the well-
documented sensory sensitivities, particularly in the tactile domain, prevalent in the ASD 
population. A total of 20 ASD individuals will be assessed pre- and post- afVNS application to 
measure short-term effects on core autistic traits and commonly co-occurring conditions.  
 
 

 
3 
Population and Recruitment 
The study population will consist of individuals diagnosed with ASD. Participants will be 
recruited through multi-faceted strategy to achieve the target sample size of 20. This includes 
active referrals from local clinical practices specializing in neurodevelopmental conditions, 
collaboration with non-governmental organizations (NGOs) serving the autistic community, 
and the use of approved study advertisements within these networks. Enrollment will be 
tracked weekly and outreach will be expanded to additional clinical partners if recruitment falls 
behind schedule. Written informed consent will be provided by all subjects or parents/ 
guardians prior to their participation. A qualified expert will assess the eligibility of each patient 
according to the inclusion and exclusion criteria as detailed below:  
 
Inclusion criteria 
• 
Written informed consent 
• 
Age: between 7 and 22 years 
• 
Participants and parent/ guardian have sufficient proficiency in English language 
• 
Diagnosis of ASD as defined by Autism Diagnostic Observation Schedule 2nd edition 
(ADOS-2) or Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-
5)  
• 
Full-Scale Intelligence Quotient (FSIQ) ³70  
 
Exclusion criteria 
• 
Severe psychiatric disorders (e.g. bipolar, major depressive disorder) 
• 
Severe neurological disorders (e.g. stroke, epilepsy) 
• 
Presence of a known pathogenic genetic variant associated with neurodevelopmental 
disorders (e.g. Fragile X, Rett Syndrome, Tuberous Sclerosis) 
• 
History of cardiovascular disease 
• 
History of head trauma (surgery or tumor) 

 
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• 
Active medical implants (cochlear, VNS or pacemakers) 
• 
Cerebral shunts 
• 
Auricular skin disease that compromises placement of electrodes 
• 
Pregnancy  
 
Patient and Public Involvement (PPI) 
This trial design was informed by clinical expert consultation. While formal PPI was not part of 
this initial protocol development, we have outlined a plan for involving the autistic community 
in the interpretation of results and future research. 
 
Outcome measures 
The primary outcome measure is feasibility measured from completion rate and 
neurostimulation tolerance of home healthcare application with successful utilization of daily 
afVNS. Participants or their caregivers will complete a daily usage log to record session 
completion, duration and any issues. This will be used to distinguish between sessions missed 
due to device-related technical problems and those missed due to participant choice or 
tolerability. This distinction is critical for interpreting the primarily feasibility endpoints and is 
detailed in the Statistical Analysis Plan (SAP).  
 
The secondary measures enable assessment of afVNS clinical and physiological effects, 
which will be measured at pre- (‘baseline’) and post- (‘final day’) afVNS (Figure 1; Table 1). 
These measures include: 
• 
Clinician Global Clinical Impression (CGI-I) for core social impairment and overall level 
of cognitive, adaptive and social functioning. 
• 
Clinician Global Impression Severity (CGI-S) for assessment of social interactions, 
restricted or repetitive behaviors and overall autistic traits. 

 
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• 
Clinician Global Impression Efficacy (CGI-E) for assessment of therapeutic 
intervention on core autistic traits and associated functional domains. 
• 
Parent-Rated Anxiety Scale for ASD (PRAS-ASD) for anxiety assessment consisting 
of 25 questions related to anxiety ranging from 0 (none) to 3 (severe). 
• 
Parent-rated Aberrant Behavior Checklist (ABC) of 58 items on 1-4 Likert scale with 
five subscales including: irritability, hyperactivity, lethargy/ withdrawal, stereotypy and 
inappropriate speech. 
• 
Parent defined target symptoms. One or two problems of greatest concern to parents 
at baseline rated on frequency, duration, intensity and functional impairment, assessed 
on a 9-point scale as 1 = normal, 2 = markedly improved, 3 = definitely improved, 4 = 
equivocally better, 5 = no change, 6 = equivocally worse, 7 = definitely worse, 8 = 
markedly worse; and 9 = disastrously worse. Ratings across the two target symptoms 
will be averaged. 
• 
Pittsburgh Sleep Quality Index (PSQI) or Cleveland Adolescent Sleep Questionnaire 
(CASQ) for assessment of sleep quality.  
• 
Controlled Oral Word Association Test (COWAT) for assessment of verbal fluency. 
• 
Autonomic activity measured from wearable sensors with continuous data collection 
for 30 mins will be collected while the subject sits in a quiet room.  
 
Study Protocol 
Outcome measures will be assessed at baseline (Day 1) and final day (Day 15) (Figure 1). At 
baseline, research subjects or their caregivers will be trained in applying the neurostimulation 
device for use of the stimulators and controller unit. Research subjects or their caregivers will 
be required to demonstrate successful device operation and electrode placement before being 
approved to participate. Thereafter, the device will be utilized at home, and research subjects 
or caregivers will self-administer daily afVNS, ideally at a consistent time each day. 
Participants will complete daily usage records online, which will be reviewed weekly by the 

 
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study coordinator to monitor adherence. Research subjects with previously prescribed drug 
medications will continue their stable treatments without change during the clinical study. No 
new neuromodulation therapies are permitted for the duration of the trial.  
 
Non-invasive VNS 
afVNS will be performed by NX-08® device (supplied by Neuropix Technologies Ltd, Hong 
Kong). NX-08® device is a wireless neurostimulator with ear electrodes positioned at the 
cymba conchae of the auricle for afVNS. Stimulation intensity (mA) for treatment will be 
determined for each subject by tolerance measures at baseline (Day 1) visit, where patients 
will be assessed for their stimulation pain threshold by application of the device. Thereafter, 
afVNS will be applied for 1 hour per session, once daily, for 14 consecutive days.  
 
Safety Monitoring and Assessment of Harms 
Adverse events (AEs) will be defined as any untoward medical occurrence in a trial participant, 
which does not necessarily have a casual relationship with the intervention. Serious Adverse 
Events (SAEs) will be defined per standard ICH-GCP criteria. Harms will be assessed 
systematically through multiple channels: (1) participant/ caregiver reporting via a daily log 
and weekly check-in calls; (2) direct questioning by the study team during their final 
assessment, and (3) review of device usage data for signs of non-use that may indicate 
tolerability issues. 

 
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Figure 1. Study design for home application of auricular fiber Vagus Nerve Stimulation (afVNS) for 
Autism Spectrum Disorder (ASD) individuals. Recruitment of research subjects (n=20) will be 
followed by pre- (Day 1) and post- (Day 15) afVNS clinical assessments. 
 
 
 
 
 
 
 
 
 

 
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Baseline Measures 
Daily Measures 
Endpoint Measures 
Subject 
compliance 
and completion 
 
Stimulation tolerance, 
successful 
deliverance, 
stimulation time and 
completion 
record; 
adverse effects; and 
picture 
of 
device 
placements. 
Stimulation 
history 
and compliance rates. 
Overall 
ASD 
– 
anxiety, 
social 
interaction  
CGI-I, CGI-S, CGI-
E, ABC 
 
CGI-I, CGI-S, CGI-E, 
ABC 
Specific 
ASD 
symptoms 
Parent 
defined 
target symptoms 
 
Parent defined target 
symptoms 
Verbal Fluency 
COWAT 
 
COWAT 
Anxiety 
PRAS-ASD 
 
PRAS-ASD 
Sleep 
PSQI or CASQ 
Wearable Tracker 
PSQI or CASQ 
Autonomic Activity 
Wearable Measure 
 
Wearable Measure 
 
Table 1. Study measures in Autism Spectrum Disorder (ASD) individuals for evaluation of clinical 
and physiological effects following auricular fiber Vagus Nerve Stimulation (afVNS). Clinician 
Global Clinical Impression (CGI-I); Clinician Global Impression Severity (CGI-S); Clinician Global 
Impression Efficacy (CGI-E); Parent-Rated Anxiety Scale for ASD (PRAS-ASD); Aberrant Behavior 
Checklist (ABC); Parent defined target symptoms; Pittsburgh Sleep Quality Index (PSQI); 
Cleveland Adolescent Sleep Questionnaire (CASQ); Controlled Oral Word Association Test 
(COWAT).            
 
 
 
 
 
 
 
 
 

 
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Recording Autonomic Nervous System (ANS) Activity 
Autonomic activity of ASD individuals will be measured and characterized pre- (‘baseline’) and 
post- (‘final day’) afVNS. Each subject will wear a heart rate sensor for 30 min for continuous 
data collection while sitting in a quiet room (Figure 2). Real-time data will be acquired, and 
autonomic indices (Table 2) will be analyzed with HRV software (Kubios HRV Scientific). 
Autonomic activity of ASD individuals will be compared to aged-match controls.        
 
 
 
 
 
Figure 2. Research subjects will sit for 30 mins in a quiet room with a wearable heart rate sensor 
attached via a chest strap for measurement of the autonomic nervous system activity. Data will be 
analyzed for key autonomic indices.    
 
 
 
 
 
 
 

 
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ANS 
Indices 
Name 
Description 
HR 
Heart Rate 
Heart beats per minute. 
HRV 
Heart Rate Variability 
The variance in time between each 
heartbeat. 
Mean 
R-R 
Intervals 
Average 
number 
of 
intervals 
between adjacent R waves 
The mean time interval between two 
consecutive heartbeats. 
NN50 
Number of pairs of successive NN 
(R-R) intervals that differ by more 
than 50 ms 
The number of interval differences of 
successive R-R intervals greater than 50 
ms. 
pNN50 
Percentage of adjacent NN (R-R 
intervals that differ by more than 50 
ms 
The proportion of NN-50 to the total of 
RR-intervals. 
sdNN 
Standard deviation of NN (R-R) 
intervals 
Standard deviation of the R-R interval. 
RMSSD 
Root mean square of successive 
differences 
Indicates high frequency influences of 
heart rate variability. 
HF Power  
 
High Frequency Power 
Indicates the respiratory sinus arrhythmia 
(RSA) – the natural variation in heart rate 
that occurs with breathing. 
LF/HF Ratio 
Low Frequency to High Frequency 
Ratio 
Indicates the index of sympathovagal 
balance. 
Poincare 
Plot SD1 
Poincare Plot Standard Deviation 1 
The standard deviation of short-term 
heart rate variability; a direct non-linear 
equivalent of HF power.  
Poincare 
Plot SD2 
Poincare Plot Standard Deviation 2 
The standard deviation of long-term heart 
rate 
variability; 
a 
direct 
non-linear 
equivalent of overall HRV. 
 
Table 2. Summary and description of the main autonomic nervous system indices across time-, 
frequency- and non-linear domains. 
 
 
 
 
 
 
 

 
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Study Oversight 
The trial sponsor serves as the coordinating centre responsible for overall trial management, 
regulatory compliance, and data analysis. The Principle Investigator holds responsibility for 
the scientific conduct at the study site. Given the pilot feasibility design, a formal steering 
committee or independent endpoint adjudication committee will not be constituted.  
 
Oversight of data and safety will be managed by the Principal Investigator and the sponsor. A 
formal independent Data Monitoring Committee (DMC) is not required due to the short 
duration, single-arm design, and the low-risk, non-invasive nature of the afVNS intervention. 
No formal interim analyses for efficacy or futility are planned. The trial may be stopped 
prematurely on grounds of safety, such as occurrence of a serious adverse event deemed 
related to the device, or for clear futility in achieving feasibility endpoints. 
 
Trial conduct will be monitored internally. The Principal Investigator and study coordinator will 
review recruitment progress, protocol adherence, and adverse events on a weekly basis. The 
sponsor will also perform periodic remote monitoring of the study database for data quality 
and completeness.  
 
Participant Retention 
To promote retention, participants will receive reminder calls/ messages before assessments. 
For participants who discontinue the intervention, outcome data will still be collected at the 
final timepoint where possible to minimize missing data. 
 
Ethics, Consent, Study Organization and Registration 
The clinical trial has been approved by the HKSTP Clinical Research Ethics Committee 
(CREC) 
IRB 
(No. 
2025-031) 
and 
is 
registered 
in 
the 
clinical 
trials 
register 
http://www.clinicaltrials.gov (NCT06473623). The study complies with the code of Ethics of the 
World Medical Association Declaration of Helsinki. The investigator will explain the benefits 

 
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and risks of participation in the study to each research subject and will provide an informed 
consent form approved by the independent Ethics Committee. Only subjects or their parents/ 
guardians, who sign the form, will be included in the study. The research data will be published 
anonymously. 
 
Data Collection and Management 
Clinical and physiological data will be collected using standardized Case Report Forms (CRFs) 
by trained assessors according to the following schedule: (a) Screening Visit: Prior to the 
initiation of afVNS, participants will be screened for eligibility, and all relevant data will be 
documented; (b) Baseline and Follow-up Visits: All study application sessions and scheduled 
follow-up assessments will be documented upon completion; (c) Adverse Event Reporting: 
Any Adverse Events (AEs) will be documented promptly on occurrence. All forms will be dated 
and signed by the principal investigator or an authorized delegate. CRFs will be monitored for 
completeness and correctness. Only complete and correct data will be entered into a 
password-protected electronic study database by the designated data manager and stored on 
a secure server. Access to study data is restricted to authorized personnel. In all cases, it 
remains the responsibility of the investigator to check that CRFs are completely filled in. 
Original CRFs will be stored in a locked cabinet at the study site. All study related data 
(electronic as well as on paper) will be stored for 3 years after trial completion.  
 
Confidentiality 
All personal information will be kept strictly confidential. Participant data will be 
pseudonymized using a unique study ID at the point of data entry. Electronic records will be 
stored on password-protected, access-controlled secure servers with regular backups. Data 
collected is for the purposes of this study only; the consent form does not include provisions 
for the use of data or biological specimens in future ancillary studies. 
 
Protocol Modifications 

 
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Any important modifications (e.g. change to eligibility criteria, outcomes, analyses) that may 
impact the study’s conduct or participant safety will require prior approval from the Ethics 
Committee. Approved amendments will be communicated to all study site personnel and will 
be updated in the clinicaltrials.gov registry. Participants will be re-consented if the modification 
directly affects their participation.  
 
Withdrawal from the study 
In case of endangerment of personal safety, intolerable discomfort from the stimulation (as 
defined by a participant request to stop), lack of compliance, or withdrawal of informed 
consent, the research subject will be excluded from further participation.  
 
The study involves a non-invasive neurostimulation device with a well-established safety 
profile. In the unlikely event of harm arising from participation in this trial, all participants are 
covered by insurance as required by local regulations. Standard clinical care will continue to 
be provided by the participant’s treating physicians; no ancillary or post-trial care is provided 
by the study team beyond the trial period. 
 
Sample Size Calculation & Statistical Analyses 
The study protocol is designed with a primary objective of evaluating feasibility and usability 
of afVNS in individuals with ASD. A sample size of 20 participants has been determined to be 
appropriate for early-phase investigation. This number aligns with established methodological 
recommendations for pilot studies, which typically enroll 10-30 participants to inform the 
design of future definitive trials. The selected sample size provides sufficient precision for 
estimations on feasibility and tolerability, yielding 95% confidence intervals of approximately 
±20% for a binary outcome, and to detect large effect sizes (Cohen’s d>0.8) in preliminary 
continuous outcomes, such as usability scores, with 80% power, a=0.05. This ‘fit-for-purpose’ 
approach is consistent with FDA guidance for early-stage device development60 and ensures 

 
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adherence to ethical principles by minimizing participant exposure, while addressing core 
questions of protocol viability. 
 
Clinical outcome data, including severity of autistic traits, will be collected at baseline and 
following completion of afVNS intervention. All statistical analyses will be performed using 
Graphpad Prism. The normality of data distribution will be assessed using Shapiro-Wilk tests. 
Based on the data type and distribution, either parametric or non-parametric tests will be 
employed. 
 
For the analysis of ANS activity, the mean value of each autonomic variable will be calculated 
from the 30 min data collection period for each subject. To compare autonomic activity 
between the ASD group and age-matched control group in a non-paired design, Welch’s t-test 
(parametric data) or Mann-Whitney U test (Wilcoxon rank-sum test, non-parametric data) will 
be used.  
 
For the primary within-subject (paired) analysis comparing pre- and post- afVNS application 
effects, pairwise statistical tests will be conducted. The paired t-test and Wilcoxon signed-rank 
test for parametric and non-parametric data will be used, respectively. These analyses will be 
applied to autonomic variables and clinical scores to evaluate the preliminary effects and 
within-subject change. Full details of statistical analyses are detailed in the SAP.