Post-acute sequelae of COVID-19 infection or Long COVID is a growing concern, even in
patients with mild initial illness. These patients develop numerous chronic debilitating
symptoms including fatigue, chest pain, reduced exercise tolerance and tachycardia, with
symptoms persisting weeks beyond the initial infection.

Preliminary data shows that post-COVID-19 POTS patients have reduced parasympathetic
(PNS) function. Given that the PNS protects against inflammation, it is hypothesize that
post-COVID-19 POTS is caused by reduced PNS activity, which in turn, contributes to
persistent inflammation, orthostatic intolerance (OI) symptoms

Aim 1 of the study is to test the hypothesis that reduced PNS activity is associated with
persistent inflammation in patients with post-COVID-19 POTS.

Aim 2: Test the hypothesis that restoring PNS function in post-COVID-19 POTS patients
with chronic transcutaneous vagus nerve stimulation (tVNS) will improve inflammation,
orthostatic tachycardia and OI symptoms.

Primary Outcome Measures:

1. To evaluate immune cell activation in post-COVID-19 POTS and patients with history
of COVID-19 infection without sequelae and correlate this with the degree of
decreased PNS activity. The primary endpoint is IL-6 levels.

2. Restoring PNS with chronic transcutaneous vagus nerve stimulation (tVNS), will
improve the symptoms of orthostatic intolerance symptoms will

Rationale:

Elevated levels of IL-6, CRP and D-dimer are found in Long COVID patients, which
resembles post-COVID tachycardia syndrome in POTS patients. Notably, stimulation of the
efferent Vagus nerve has been shown to reduce cytokine production and systemic
inflammation in response to endotoxin. Hence, decreased PNS function, as reported with
acute SARS-CoV-2 infection and in post-COVID-19 POTS patients, may render these patients
prone to persistent inflammation. The study aims to determine the link between PNS
activity and immune activation in post-COVID-19 POTS.

Study population: Total of 60 participants, 30 post-COVID-19 POTS patients and 30
controls with history of COVID-19 infection without sequelae.

Study Visits: 5 visits, 3 in person and 1 telemedicine, Study procedures include EKG,
urine and blood sample collection, Orthostatic Standing Test, Measurement of blood volume
using carbon monoxide rebreathing technique, Tilt table test.

Participants will be asked to complete an autonomic symptoms assessment questionnaire
(COMPASS-31),32 quality of life EQ-5D and neuropsychological tests that includes
Cambridge Brain sciences: Web based cognitive assessment platform and PROMIS scale:
Functional Activities Questionnaire in Older Adults with Dementia.

Transcutaneous Vagus Nerve Stimulation (tVNS): The device, which is FDA-approved TENS
7000 will be supplemented with ear clip electrodes, and the tragus or earlobe will be
used as sites for vagal stimulation. The device will be used for 30 mins, twice a day for
28 days.

Randomization: The subjects will randomized to any one of the two sites of stimulation on
the ear. There will be 50% chance to be allocated to one of the sites that may have less
stimulation. The risk of having side effects is the same for both stimulation sites.

Data and Safety Monitoring Plan: The Data and Safety Monitoring Officer (DSMO) will
provide objective review of treatment results as they relate to human safety and data
quality. Dr. Cheryl Laffer, Professor of Medicine, Division of Clinical Pharmacology will
serve as DSMO. The DSMO will review the initial protocol and will receive reports of the
progress of the study every 12 months. These reports will provide information regarding
recruiting, safety reporting, data quality, and efficacy.

Statistical Considerations:

Biostatistical Section Power analysis: The primary endpoint is serum IL-6. The proposed
sample size of 60 (30 pairs of patients and controls) provides 90% power to detect an
effect size of 0.62 for the mean difference in IL-6 between post-COVID-19 POTS (cases)
and controls (COVID-19 infected w/o sequelae), with the two-sided type I error = 5%. This
calculation is based on the preliminary data of mean difference of IL-6 ≈ 1.82 and the SD
≈ 2.94 in POTS.6 The effect size is defined as the ratio of mean IL-6 difference between
cases and controls to standard deviation.

Data analysis plan: Demographic information will be tabulated. Descriptive statistics,
including means, standard deviations, and ranges for continuous parameters, as well as
percent and frequencies for categorical parameters, will be presented. T-test or
Mann-Whitney (as appropriate) will be applied to examine the mean differences between
cases and controls with respect to the outcomes. The conditional logistic regression
model will be applied for the multivariable data analysis. The adjusted p-values and the
adjusted 95% confidence intervals (CIs) will be reported.