Cranial Electrotherapy Stimulation (CES) Influences on Acute Stress Responses
Phase: NA
Conditions: Anxiety State
Sponsor: Tufts University

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Cranial Electrotherapy Stimulation on Acute Stress  
 
 
 
 
Study Protocol 
Document version: February 24, 2023 
IRB Approval date: April 19, 2023 
 
 

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PROTOCOL TITLE: 
Cranial Electrotherapy Stimulation (CES) Influences on Acute Stress Responses 
 
PRINCIPAL INVESTIGATOR (PI): 
Name: Kana Okano 
Department: Center for Applied Brain and Cognitive Sciences 
Are you a student? Yes 
  
No 
 
   If Yes, you must designate a Faculty Advisor below. 
 
FACULTY ADVISOR (required for Student PIs): 
Name: N/A 
Department: N/A 
 
VERSION NUMBER/DATE 
 
Version 1/ February 24 2023 
 

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1.0 
Purpose of the study: 
 
The purpose of this study is to quantify the effects of 20 sessions of (Active vs Sham) 
Cranial Electrotherapy Stimulation (CES) on multiple measures of acute stress 
responses: biochemical (salivary alpha amylase and cortisol), physiological (e.g., heart 
rate, heart rate variability, respiration rate), emotional (state anxiety), and behavioral 
(i.e., cognitive task performance). 
 
2.0 
Background / Literature Review / Rationale for the study: 
 
Acute Stress 
Soldiers are continually exposed to acute stressors during training and operations, 
activating a cascade of biochemical changes in the body, including an initial 
sympathetic-adrenal medulla (SAM) catecholamine-related response, and a slower-
moving hypothalamic-pituitary-adrenal (HPA) glucocorticoid-related response (Angelova 
et al., 2021; Axelrod & Reisine, 1984; Buchheim et al., 2019; Charmandari et al., 2005; 
Gagnon & Wagner, 2016; McEwen, 2007; Padgett & Glaser, 2003). The diverse effects 
of acute stress on neurotransmitters and the central nervous system are associated with 
similarly diverse perceptual and cognitive effects, generally related to the intensity of 
experienced stress and the processes demanded by specific mental tasks. For example, 
mild-to-moderate acute stress can improve performance on relatively basic (or well-
learned and rehearsed) perceptual and cognitive tasks, such as simple reaction time and 
verbal memory retrieval, perhaps owing to activation of the ascending reticular activating 
system (ARAS) (Arnsten, 2009; Broadbent, 1971; Brunyé et al., 2021a, 2021b; De Cicco 
et al., 2018; Hupbach & Fieman, 2012; Shields et al., 2019). Moderate-to-high acute 
stress, in contrast, can negatively influence performance on tasks more demanding of 
executive processes and spatial memory retrieval, perhaps owing to HPA-related 
activation and brain regions (e.g., prefrontal cortex, hippocampus) with high 
glucocorticoid receptor densities (Arnsten, 1998, 2009; Brunyé et al., 2016, 2019; 
Cerqueira et al., 2007; Luine et al., 1994; Olver et al., 2015; Richardson & VanderKaay 
Tomasulo, 2011; Shields et al., 2015, 2016). 
 
Individuals engaged in high-stakes occupations, such as military personnel, first 
responders, and emergency medicine physicians, are continually exposed to moderate-
to-high stress, which can negatively influence their ability to sustain performance on job-
related duties (Meredith et al., 2011; Orasanu & Backer, 1996; Reynolds & Wagner, 
2007; Sood et al., 2011). As a result, scientists and practitioners alike seek methods to 
reduce the stress response and increase resilience to its short- and long-term effects. 
This includes efforts to train emotion regulation strategies, resilience, mindfulness, and 
biofeedback (Berking et al., 2010; Jha et al., 2015; Kennedy & Parker, 2019; Reivich et 
al., 2011). Methods also include non-invasive brain stimulation, including transcranial 
direct current stimulation (tDCS), transcranial alternating current stimulation (tACS), 
transcranial magnetic stimulation (TMS), and cranial electrotherapy stimulation (CES). 
Each of these techniques has received attention for its potential utility in altering 
biochemical and physiological manifestations of acute stress responses and thus 
altering behavior. 
 
Cranial Electrotherapy Stimulation 
Cranial electrotherapy stimulation (CES) differs from transcranial electrical stimulation 
(tES) in several ways. First, CES electrodes include one anode and cathode but are 

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typically attached to the temples, mastoids, or earlobes (rather than the scalp). Second, 
CES is typically administered using alternating rather than direct current, at relatively low 
intensities ranging from 50-500 µA and frequencies ranging from 0.5 to 100 Hz. Third, 
rather than targeting specific brain regions, CES is thought to exert nonspecific effects 
on both the central and peripheral nervous systems. Many mechanistic explanations 
regarding CES effects on the central and peripheral nervous systems, neurotransmitters 
and hormones, and emotion and behavior have been proposed (see Brunyé et al., 
2021a, 2021b). Regarding central and peripheral nervous system effects, various 
publications have proposed that CES influences sympathetic versus parasympathetic 
drive, alters oscillatory brain activity, increases cerebral blood flow in subcortical brain 
regions, stimulates afferent projections of the vagus and/or trigeminal nerve, and 
modulates functional brain connectivity and plasticity. Regarding neurotransmitter and 
hormonal effects, studies have variably proposed that CES alters SAM and HPA axis 
and associated neurotransmitters such as cortisol, serotonin, norepinephrine, dopamine, 
acetylcholine, and ϒ-aminobutyric acid (GABA) (Barclay & Barclay, 2014; Datta et al., 
2013; Ferdjallah et al., 1996; Kavirajan et al., 2014; Lee et al., 2019; McClure et al., 
2015; Roh & So, 2017; Schroeder & Barr, 2001; Wagenseil et al., 2018; Winick, 1999; 
Yennurajalingam et al., 2018). Unfortunately, no comprehensive model of CES provides 
mechanistic linkages between these putative effects of CES or provides compelling 
associations to behavioral outcomes. 
 
According to computational models of CES current propagation, CES has the strongest 
neuromodulatory effects on diverse cortical and subcortical brain regions including the 
temporal lobes, medulla oblongata, midbrain, thalamus, hypothalamus, pons, and insula 
(Datta et al., 2013; Ferdjallah et al., 1996). In research examining brain activity with 
electroencephalography (EEG) during and after CES administration, some studies find 
evidence for alpha and beta band power modulation, suggesting that CES may be 
expected to influence arousal states and attention (Lee et al., 2019; Schroeder & Barr, 
2001; Wagenseil et al., 2018). Finally, two studies have examined brain hemodynamics 
during and after CES administration, using both functional magnetic resonance imaging 
(fMRI) and xenon-enhanced computed tomography (XeCT). In the fMRI study, the 
authors found that CES induced broad regional brain deactivation and altered default 
mode network activity and proposed that CES may induce transient downregulation of 
rumination or worry (Hamilton et al., 2011). In the XeCT study, the authors found that 
CES altered cerebral blood flow (CBF) specifically in the brainstem and thalamus, 
suggesting that CES plays a role in modulating anxiety and pain perception (Gense de 
Beaufort et al., 2012). 
 
In addition to its effects on functional brain activity, CES has also been found to 
modulate salivary, urinary, blood, or cerebrospinal fluid (CSF) levels of stress hormones, 
inflammation and immune markers, and neurotrophic factors. These studies find highly 
mixed results. For example, while most studies suggest that CES does not influence 
serotonin, dopamine, β-endorphins, alpha amylase, cortisol, adrenocorticotrophic 
hormone (ACTH), C-reactive protein, interleukin-1, or interleukin-6 (Cho et al., 2016; 
Krupitsky et al., 1991; Roh & So, 2017; Yennurajalingam et al., 2018), an earlier study 
suggested that CES administration decreases cortisol and increases ACTH, serotonin, 
and β-endorphins (Liss & Liss, 1996). Note that the latter study shows a high risk of bias, 
with experimenters testing themselves as participants, financial conflicts of interest, and 
a lack of random assignment or blinding to CES conditions (Brunyé et al., 2021a, 2021b; 
Kavirajan et al., 2014). 
 

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In clinical contexts, CES devices are classified as class II (special controls) medical 
devices for the treatment of anxiety or insomnia and as class III medical devices for the 
treatment of depression (Docket No. FDA-2014-N-1209). In one of the largest placebo-
controlled CES studies to date, active (versus Sham) CES (100 µA, 0.5 Hz, 35 days of 
treatment) was administered to 115 participants with an anxiety disorder; results showed 
an approximately 32% reduction in anxiety measured using the Hamilton Rating Scale 
for Anxiety (HAM-A-17) (Barclay & Barclay, 2014). However, results are generally mixed 
about CES effectiveness in treating clinical insomnia (Feighner et al., 1973; Lande & 
Gragnani, 2013; Wagenseil et al., 2018; Weiss, 1973) and depression (Kavirajan et al., 
2014; McClure et al., 2015; Mischoulon et al., 2015).  
 
In our own recent study (Brunye et al., 2022), we used a double-blind, placebo-
controlled, crossover design to examine the effects of CES on emotional, physiological, 
biochemical, and behavioral responses to acute stress. Healthy male participants visited 
the laboratory for two sessions, one involving active CES administration (100 µA, 0.5 Hz, 
20 min administration) and one involving Sham, inactive CES. During each session, 
participants were placed under stress (threat of torso shock) while performing 
challenging cognitive tests, and we measured emotional, biochemical (alpha amylase, 
cortisol), physiological (heart rate, respiration rate, heart rate variability, pupil diameter), 
and cognitive behavioral (memory, decision-making, spatial orienting) responses. 
Outcome metrics were compared using repeated measures analysis of variance 
(ANOVA) and planned comparisons. The stress induction reliably modulated measures 
of sympathetic adrenal medullary (SAM) activity but not hypothalamic–pituitary–adrenal 
(HPA) axis activity. Active versus placebo CES did not significantly influence any 
emotional, biochemical, or physiological outcome measure; there was, however a 
consistent numerical pattern wherein active CES did appear to reduce some of the 
physiological responses to stress relative to Sham CES. Furthermore, active CES did 
selectively increase performance on a recognition memory test and degrade 
performance on a perceptual decision-making test; however, overall performance on our 
cognitive tasks was very low, suggesting a potential floor effect that could be masking 
any additional effects of CES. Overall, our study found very limited evidence that CES 
reliably modulates the acute stress response. 
 
The Present Study 
The present study is designed to extend our recent study in three primary ways.  
 
First, rather than a fixed intensity of CES (100µA), we will use a standardized procedure 
to identify each participant’s sensory threshold (Bystritsky et al., 2008; Gong et al., 2016; 
Schroeder & Barr, 2001). Because 100µA was consistently sub-threshold (i.e., 
imperceptible) in our prior participants, it is likely this procedure will result in higher (e.g., 
250-500µA) CES intensities administered. There are three primary reasons why we have 
chosen to use higher intensity CES, and why we believe it will make it more likely to find 
CES effects on physiology, biochemistry, and behavior: 
1. Two extant research studies suggest that 300µA (Bystritsky et al., 2008; 
Overcash et al., 1989) and up to 1500µA (Smith et al., 1994) are effective at 
lowering anxiety, in line with the conclusions of a review examining CES 
effects on anxiety and stress (De Felice, 1997). 
2. Most of the data from our prior research with the device shows the predicted 
pattern of results, for example lower heart rate variability and respiration rate 
with CES (vs sham); however, the pattern is numerical only and does not 
reach traditional significance levels. We believe that higher intensity CES may 

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amplify these previously noted patterns; of course, this is an empirical 
question and cannot be answered without additional data collection.  
3. This procedure of individualized thresholding is congruent with manufacturer’s 
recommendations, and they report seeing the most reliable anxiolytic-like 
effects with this procedure. Also, the procedure of individualized thresholding 
and minimum intensity (≥250µA) is congruent with the Special Operations 
Command’s (SOCOM) current use of the device during military training and 
operations, where they report anecdotal success for stress mitigation.  
 
Second, rather than a single 20-minute session of CES administration, we will use daily 
administration (20-minutes/day) over the course of 4-6 weeks.  
 
These first two modifications are being made to better conform with the device 
manufacturer’s (Electromedical Products International, Inc., Mineral Wells, TX) 
recommendations for CES administration.  
 
Finally, to elicit a more robust stress response that activates both SAM and HPA activity, 
we will increase the intensity (to level 3 of 5) of our torso shock administration. With 
these changes, we make four primary hypotheses: 
 
 
H1: Biochemistry: We hypothesize that Active vs Sham CES will reduce 
biochemical responses to stress, including salivary alpha amylase as an indicator of 
SAM activity and salivary cortisol as an indicator of HPA activity. 
 
H2: Physiology: We hypothesize that Active vs Sham CES will reduce 
physiological responses to stress, including heart rate, heart rate variability, respiration 
rate, and pupil diameter.  
 
H3: Emotion: We hypothesize that Active vs Sham CES will reduce subjective 
experiences of stress, including responses on the STAI. 
 
H4: Behavior: We hypothesize that Active vs Sham CES will lead to increased 
performance on tasks typically degraded by stress exposure, including a recognition 
memory test and a spatial memory test. 
 
3.0 
Participant Population: 
 
We expect to recruit up to 500 individuals, including Soldiers and civilians, to achieve a 
final sample size of 40 complete data sets. Our high recruitment number allows for 
Soldier group briefings (active-duty units at their home station and HRV cohorts at 
DEVCOM SC) which are routinely 30+ and can be upwards of 100 Soldiers.  
 
We anticipate an approximately 40-50% attrition rate given our requirement to meet a 
minimum CES threshold of 300µA (i.e., a 250µA minimum CES intensity) and the 
prolonged study duration. To accommodate the potentially high attrition rate, we expect 
to recruit and consent up to 500 Soldiers and/or civilians, and schedule and enroll up to 
100 Soldiers and/or civilians. 
 
We are recruiting civilians in addition to military personnel because we have limited 
access to military personnel to conduct this research, and we have no a priori reason to 
believe that civilians will show markedly different stress responses, task performance, or 
CES responses relative to military personnel. In fact, most extant research examining 
the utility of CES for stress reduction is conducted with civilians. Because one of our 
tasks (marksmanship) could be considered geared towards military personnel, see 

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Section 15 for a description of possibly increased risk of psychological harm to civilians 
versus military personnel. 
We will recruit participants 18-40 years of age, or 17 years if emancipated minor. 
This age range was chosen to represent the majority of Active Duty enlisted 
military personnel, which is under 41 years of age (Defense, 2014). There is no 
gender-based enrollment restriction. 
Inclusion Criteria: Eligible individuals include those who:  
 
1. Can sit and stand freely. 
2. Have not used or experienced CES administration in the past. 
3. Agree to have their data stored in a repository (database) for future use. Peer-
reviewed scientific journal may require data to be stored in a repository as a 
condition for publication. Such repositories are online databases, in which coded 
data is linked (no link to participants personally) to the journal article citation. 
Exclusion Criteria: The following exclusion criteria will be evaluated by self-report (on the 
Informed Consent Form) prior to beginning the study.  
 
1. Use of prescription medications, other than oral contraceptives, as they may alter 
stress hormones (Granger, Hibel, Fortunato, & Kapelewski, 2009). 
2. Women only: 
a. Pregnant or plan to become pregnant during the study, as the shock belt  
may be unsafe for pregnant women and their children. 
b. Nursing, as it may alter stress hormones (Ahn & Corwin, 2015). 
3. History of:  
a. A neurological or psychological disorder (such as depression, anxiety 
disorders, migraines, cluster headaches, seizures, Post-traumatic stress 
disorder (PTSD) or panic attacks). Individuals with neurological and 
psychological disorders may exhibit altered stress responses, not 
representative of the population (Spijker & van Rossum, 2012). 
b. Cardiac disease (including arrhythmia or fast or skipped heart beats). The 
shock belt may be unsafe for individuals with cardiac disease (Steptoe & 
Kivimäki, 2012) 
c. Implanted medical devices, such as pacemakers. The Shock belt may be 
unsafe for individuals with an implanted medical device. 
d. Hypertension. The Shock belt may be unsafe for individuals with 
diagnosed hypertension (Spruill, 2010). This risk is low, and the 
prevalence of undiagnosed hypertension in our targeted cohort (ages 18-
40) is also very low; for these reasons, we believe self-report of 
diagnosed hypertension is sufficient. 
e. Insomnia, as insomnia is associated with chronic physiological arousal 
(Bonnet & Arand, 2010)  
f. 
Head injury (including neurosurgery, concussion, TBI, skull fracture, 
hematoma) 
g. Illness that caused brain injury 
h. Any other brain-related condition (such as traumatic brain injury) 

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i. 
Metal in the head (outside of mouth, such as shrapnel, surgical clips, or 
fragments from welding or metalwork) 
j. 
Implanted medical device (e.g., pacemaker, insulin pump) 
Study Withdrawal Criteria:  
We have five study withdrawal criteria, related to minimum CES intensities, 
failing to meet minimum performance during the criterial learning tasks, 
intolerance for torso shock, self-reported symptoms on the Daily Participant 
Questionnaire (Appendix A), and intolerance for CES. 
First, during the individualized baselining phase (part of the Thresholding and 
Baselining session), if a participant does not reach our minimum CES intensity of 
250µA (i.e., a 300µA minimum threshold), they will be dismissed from the study. 
Second, during the baseline or follow-up session, if a participant cannot reach the 
accuracy criterion during the criterial learning tasks within 5 study-test cycles, they will 
be dismissed from the study. 
Third, during the baseline or follow-up session, if a participant cannot tolerate the 
torso shock (psychologically or physically), we will reschedule their session for a 
different day. If during two consecutive sessions they cannot tolerate the level 3 
intensity, they will be dismissed from the study.  
Fourth, at the beginning of any CES session, if the participant answers a 3 or 
higher (on the scale from 1-4) to any of the Daily Participant Questionnaire items, 
their session will be rescheduled for a different day. If they report 3 or higher for 
any of these questions for two consecutive sessions, they will be dismissed from 
the study. 
Fifth, during any CES session, if a participant cannot tolerate the CES administration at 
their individualized intensity (i.e., answering ≥ 3 to a single item on the “presence and 
intensity” and “relation to stimulation” sections), they will receive secondary CES 
stimulation as described in the Procedures section (6.0). If their reported symptoms do 
not successfully subside (i.e., falling below a 3 rating) after the secondary stimulation, 
and this occurs for two consecutive sessions, they will be dismissed from the study.  
 
Each of these study dismissal criteria are described in more detail in the Procedures 
section (6.0), and detailed in the Appendix E flowchart. 
 
4.0 
Special Populations: 
 
Children
Fetuses/Neonates 
Prisoners
Members of the military 
Non-English speakers
Those unable to read (illiterate)
Employees of the researcher 
Students of the researcher

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Adults lacking capacity to consent and/or adults with diminished capacity 
to consent, including, but not limited to, those with acute medical 
conditions, psychiatric disorders, neurologic disorders, developmental 
disorders, and behavioral disorders
 
Disadvantaged in the distribution of social goods and services such as 
income, housing, or healthcare
 
Fear of negative consequences for not participating in the research (e.g. 
institutionalization, deportation, disclosure of stigmatizing behavior)
 
Approached for participation in research during a stressful situation such 
as emergency room setting, childbirth (labor), etc.
 
5.0 
Research Locations and Sample Size: 
 
5.1 Research Locations 
 
Research will be conducted at two indoor, climate-controlled locations: 
 
1. Center for Applied Brain and Cognitive Sciences (CABCS), Tufts University, 177 
College Ave, Medford, MA. 
AND  
2. U.S. Army DEVCOM Soldier Center, 10 General Greene Ave., Natick, MA 01760 
 
5.2 Sample Size 
 
Sample size estimation was based on effect sizes from Barclay & Barclay (2014) who 
found that CES reduced subjective anxiety with an effect size of f  = 0.47. Using 
GPower, the total sample size was estimated to be 38 to achieve power of 0.80 with an 
alpha criterion of 0.05. Our goal is therefore 40 complete data sets, with 20 participants 
in each of the two groups. 
 
6.0 
Procedures Involved: 
 
We will use a double-blind, placebo-controlled design with random assignment of 
participants to one of two experimental groups: Active versus Sham CES. Two versions 
of the criterial learning and virtual reality tasks have been developed (Brunyé & Giles, 
2023; used in DEVCOM Armament Center IRB protocol 18-007, Tufts University 
protocol 1808016), and will be used herein, in balanced order across participants.  
 
Military participants will be transported to and from the CABCS research performance 
site in Medford, MA, for participating in the baseline and follow-up session. If overnight 
accommodations are required for Soldiers, those will be provided along with other travel 
related items (e.g., per diem). Travel will be planned by the research team and 
coordinated with unit leadership (if troop request) or HRVPO (if HRVs). Civilian 
participants will not be provided with transportation, accommodations, or any other 
travel-related compensation. 
 
All non-disposable devices used in the protocol will be disinfected in accordance with 
Occupational Health and Safety Administration and Centers for Disease Control 

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guidance, and product label instructions, paying attention to the required personal 
protective equipment and the contact time needed to achieve disinfection. In addition, all 
frequently touched surfaces in the research area, such as workstations, countertops, 
and doorknobs will be cleaned between participants. 
 
Upon scheduling, participants will be randomly assigned to the Active or Sham CES 
condition, using the [=rand()] function in Microsoft Excel. All participants will complete a 
Thresholding & Baseline session, the CES administration sessions, and then a Follow-
up session. 
 
Testing Day 1: Thresholding & Baselining 
 
The baseline session is designed to establish each participant’s CES individualized 
threshold and their baseline stress responsiveness and performance in our virtual reality 
scenario. This session will always take place at a consistent time of day for individual 
participants (e.g., morning or afternoon), and always at the CABCS in Medford, MA, 
using the Soldier and Small Unit Ambulatory Virtual Environment (SUAVE) facility. 
 
Participants will first complete the demographics form, daily participant questionnaire, 
and emotional state (STAI-S, STAI-T; see Appendix A) questionnaires. 
 
Because our individualized thresholding procedure may result in immediate exclusion 
from the study, participants will do it prior to baselining.   
 
CES Individualized Thresholding: Participants will be asked to sit and relax in a chair, 
and we will assist them in donning the CES electrodes on the Alpha-Stim AID device 
(see Appendix D for specifications). The device has two electrode clips that attach to the 
left and right earlobes; each clip has two disposable pads that will be soaked with saline 
(to promote conductivity between the electrodes and skin). A single CES device labeled 
THRESHOLDING will always be used for this phase; this device will always be active. 
The device will be powered on and set to 0.5Hz at the lowest possible intensity (50µA). 
Participants will be told that we will increase the intensity by a very small amount every 
30 seconds and ask them to answer each of the questions on the CES side effect 
questionnaire. At each 30-second interval, the experimenter will increase the stimulation 
intensity by 50µA. 
 
If a participant has at least one symptom at 3 or higher (i.e., ≥ 3 in the Presence and 
Intensity section), and they believe it is “possibly” or “definitely” related to the stimulation 
(i.e., ≥ 3 in the Relation to Stimulation section), we will immediately stop the thresholding 
procedure. The stimulation intensity achieved will then be recorded by the experimenter. 
 
During all subsequent CES sessions, 50µA below a participant’s stimulation threshold 
will be used as that participant’s individualized CES intensity. For example, if the 
participant achieves 350µA, then all subsequent CES sessions will use a 300µA 
intensity. If the participant never reports any symptom and relation to CES as ≥ 3, the 
device will be used at the maximum intensity of 500µA. Because this study intends to 
increase stimulation intensity beyond that of our prior study, any participant who does 
not achieve an individualized stimulation intensity of at least 250µA (i.e., ≥ 300µA 
threshold) will be dismissed from the study. 
 

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Baselining. Participants who achieve the minimum 300µA threshold will then complete a 
series of criterial learning tasks, then a series of cognitive tests while under threat of 
torso shock. Prior to, during, and after these phases, participants will provide saliva 
samples and complete the STAI-S questionnaire. These phases are detailed below: 
 
1. Criterial Learning: Participants will learn and be tested to criterion on a series of 
experimental stimuli. These include a map of a fictitious virtual city, a list of 
suspicious items from a be-on-the-lookout (BOLO) list, and camouflage patterns 
or objects (e.g., things that are carried in the hands, such as a rifle, broomstick, 
pipe, etc) representative of friendly versus enemy forces. Participants will be 
required to reach a minimum of 80% accuracy on these three criterial learning 
tasks, and will repeat the study-test sequence until they reach that criterion. We 
will probe for confidence (e.g., how confident are you in your answer, on a scale 
from 1-7?) and metacognitive ratings (e.g., how likely are you to be able to point 
to the theater when standing at the restaurant, on a scale from 1-7?) after each 
study trial. See Appendix C for details on these three criterial learning tasks. In 
our prior study, all participants reached the 80% criterion for all tasks within 3 
study-test cycles; this is expected to take about 30-35 minutes. If a participant 
cannot reach criterion within 5 study-test cycles, they will be dismissed from the 
study. 
 
2. Cognitive Testing Under Stress: Participants will be brought into the virtual reality 
system and will stand in front of a large array of visual displays with surround 
sound and a vibrating platform (for multi-sensory immersion). Study personnel 
will then help the participant don the shock belt, bioharness, and eye tracking 
glasses (see Appendix D for details on these devices). The CES device will not 
be donned. After a brief eye tracker and weapon aim calibration, a 
practice/familiarization phase, and confirming that all devices are successfully 
communicating data, three cognitive tests will be performed in an interleaved 
manner. First, a recognition memory test will involve recognizing previously 
learned suspicious targets in the virtual environment while ignoring visually 
similar distractor objects; participants respond with a simulation rifle (see 
Appendix D) and an attached game controller. Second, a spatial orienting task 
that involves pointing towards a series of distant (out of visible range) target 
landmarks (e.g., bank, theater) in the virtual environment and estimating distance 
to those landmarks; participants reorient themselves and select a direction by 
using the simulation rifle and attached game controller.  
 
Finally, a decision-making task that involves a simulated entry control point: 
participants will judge whether an approaching person is friendly, or enemy 
based upon their camouflage pattern and/or objects being carried, and use this 
information to either let them pass or fire upon them (using the rifle). During this 
task, there may be one (i.e., low workload) or many (i.e., high workload) potential 
targets simultaneously appearing in the scene. Participants will also provide 
confidence ratings for their decisions during these tasks. During task 
performance, we will use remote TRACKPACK/E infrared cameras (ART, GmbH, 
Germany) to continuously track and log weapon position, head and eye gaze 
position, and physiological measures of the bioharness. Depending upon which 
rifle is used for the study (based on availability and functionality), it may or may 

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not also collect inertial measurement unit (IMU) data with an integrated sensor 
(i.e., roll, pitch, yaw, xyz position).  
During the cognitive testing, if an incorrect response is provided by participants 
(e.g., > 45˚ absolute angular error during the spatial orienting task, incorrectly 
firing upon a friendly or incorrectly letting an enemy pass during the decision-
making test), the system will automatically trigger a torso shock. The shock belt 
will be set at level 3, which (in our previous studies) participants tend to find 
uncomfortable and stressful, but not painful. If participants cannot tolerate the 
shock and ask for it to be disabled, we will pause data collection and give them a 
5-minute rest period and ask whether they would like to try it again. If they 
respond yes, we will resume data collection (and repeat this rest-retry procedure 
as necessary). If they respond no, we will ask whether they are willing to be 
rescheduled and try again on another day; if they respond no, we will not 
reschedule. If they respond yes, we will reschedule the participant for another 
session. If during two consecutive sessions they cannot tolerate the shock, they 
will be dismissed from the study.  
Upon completion of testing, participants will return to the private seating area, 
where they will complete the additional saliva sampling and STAI-S. 
 
3. Saliva Sampling: Participants will provide saliva samples using the Salivabio Oral 
Swab (SOS) method from Salimetrics. This sampling method involves 
participants placing a hygienic absorbent swab under their tongue for 2 minutes, 
and then placing the swab into a vial. The advantage of the SOS method is that 
participants can do it entirely independently (opening the swab package, placing 
the swab under the tongue, removing swab from mouth, placing swab in the vial, 
closing the vial) and without direct contact from the experimenter. Using this 
method, saliva samples will be taken immediately prior to Questionnaires, 
immediately prior to Cognitive Testing Under Stress, immediately following 
Cognitive Testing Under Stress (accompanied by emotional state 
questionnaires), and at 20-minutes, 40-minutes, and 60-minutes after. In this 
manner, we will collect a total of 5 saliva samples during this Baseline session. 
After completing this series of saliva samples, questionnaires, and tasks, participants will 
be briefed on the CES session procedures. Participants will then be released for the day. 
Overall, we expect the Baseline session to last approximately 3 hours, as detailed in the 
below example schedule: 
0800-0815 
Questionnaires (daily checklist, STAI-S/T, demographics) 
0815-0820
CES Thresholding 
0820-0825 
Saliva Sampling 
0825-0900
Criterial Learning 
0900-0920
Don Bioharness & Shock Belt; Don & Calibrate Eye Tracking 
Glasses & Rifle
0920-0945
Cognitive Testing Under Stress 
0945-0950 
Saliva Sampling & STAI-S 
1005-1010
Saliva Sampling & STAI-S 
1025-1030 
Saliva Sampling & STAI-S 

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1045-1050
Saliva Sampling & STAI-S 
1050-1100 
CES Session Familiarization 
1100
Dismissal 
 
 
 
 
The CES Sessions 
 
Participants will then return to the laboratory at CABCS or visit the laboratory at the 
DEVCOM Soldier Center on a daily basis (Monday through Friday) for up to six 
consecutive weeks, or until they achieve a total of 20 sessions (whichever occurs first). 
They will be scheduled at a semi-consistent time every day (i.e., in morning or 
afternoon), and complete a session expected to last about 30-60 minutes. During each 
session, participants will complete the daily participant questionnaire, the STAI-S, a CES 
stimulation period involving Active or Sham stimulation, a CES secondary administration 
(as needed), and the STAI-S.  
 
If a participant answers 3 or higher on any question on the daily participant 
questionnaire, they will be rescheduled for a different day. If they report 3 or higher on 
any of the questions for two consecutive sessions, they will be withdrawn from the study.  
 
1. Active or Sham CES Administration: Participants will be asked to continue sitting 
and relaxing in the chair and the electrode cable (still attached to their earlobes) 
will be connected to a different CES device. Two types of CES devices will be 
used: an Active device or a Sham device. The Sham devices are manufactured 
by EPII and look and behave exactly like the Active devices, however they do not 
actually send any electrical current to the electrodes. The two versions of the 
devices will be coded as devices A and B, and neither the experimenter nor 
participant will know which is Active and which is Sham; only the Principal 
Investigator will keep the key to decode whether the device is Active or Sham, to 
be revealed only during data analysis (or to a medical professional if there is an 
adverse event). The experimenters and participants will only know which coded 
device (device A or B) to use during CES sessions. In both conditions, the device 
will be powered on and set to the intensity identified during the CES 
Individualized Thresholding phase, and a countdown timer (displayed on the 
device screen) will begin.  
 
After the 20 minutes of stimulation have elapsed, the device will automatically 
turn off. Immediately after the 20-minute CES administration, participants will 
complete the STAI-S questionnaire a second time, and the CES side effects 
questionnaire.  
 
2. CES Secondary Administration: If a participant answer is ≥3 to one or more 
questions on the CES side effects questionnaire (in both the “presence and 
intensity” and the “relation to stimulation” sections), we will follow the 
manufacturer’s guidelines to administer additional CES until symptoms subside. 
According to the manufacturer’s internal research (derived from an analysis of 
over 10,000 CES sessions), brief secondary CES administrations can help 

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alleviate most side effects in most circumstances. Specifically, we will provide 
successive 5-minute secondary CES administrations at the individualized 
intensity. After each secondary CES administration, there will be a 1-minute 
break when the participant will again complete the CES side effects 
questionnaire. The successive 5-minute CES administrations with interleaved 
CES side effects questionnaire will be repeated up to twice, or until the negative 
sensations subside (i.e., all ratings ≤ 3), whichever occurs first. The number of 
secondary CES administrations necessary to resolve vestibular sensations or 
anxiety (i.e., 1 or 2) will be recorded by the experimenter. If the participant’s 
reported symptoms do not subside with secondary CES administration, and this 
occurs for two consecutive CES sessions, they will be withdrawn from the study. 
Participants will then be dismissed until their next session. 
Below is an example schedule for a CES session:     
0800-0810
Questionnaires (daily checklist, STAI-S) 
0810-0830 
CES Administration 
0830-0835
Side Effects Questionnaire 
0835-0850 
Secondary CES Administration (as necessary) 
0850-0900
STAI-S Questionnaire 
0900
Dismissal 
 
Our goal is to have each participant complete a total of 20 CES sessions, though we do 
expect some attrition (see Section 3.0). We will prioritize the number of sessions (i.e., 
reaching 20) over the duration of the study; for example, some participants may 
complete the 20 sessions study in a 5-week period (rather than 4-week period) due to 
scheduling issues. We will allow up to 6 weeks to complete the study; if the 20 sessions 
are not completed in 6 weeks, the participant will be removed from the study. Variability 
in completion time (i.e., inter-session lag) will be recorded and used as a covariate in 
statistical analyses. 
 
At the end of the final CES session (session 20), participants will complete the Final 
CES Session Questionnaire (Appendix A) to assess whether our blinding was 
successful, and their opinions about CES. 
 
Final Testing Day: Follow-up 
 
The final testing day will serve as the follow-up session to assess whether 20 sessions 
of Active (versus Sham) CES will impart any changes to the biochemical, physiological, 
affective, or behavioral responses to stress induction. This session will be held at 
approximately the same time of day as the thresholding and baselining session, within 5 
days of the last daily CES session. The procedures for this session will precisely match 
the thresholding and baselining session but without the thresholding phase. After 
completing the final testing day, participants will have completed the study. 
 
Data Analysis 
 

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We will analyze data from our biochemical, physiological, affective, and behavioral 
measures using Generalized Estimating Equations (GEEs) and/or Analyses of Variance 
(ANOVAs) to examine changes over the 20 CES sessions, changes from baseline to 
follow-up, and changes due to Active versus Sham CES. An effect will be deemed 
statistically significant if the likelihood of its occurrence by chance is p < 0.05; to reduce 
the likelihood of a Type I error, any follow-up comparisons (e.g., t-tests) will use a 
Bonferroni-corrected alpha criterion. 
 
6.1 
Additional Safeguards for Special Populations:  
 
This research is funded by the Department of Defense, and the funding agency is 
interested in how military personnel respond to CES administration; for that reason, we 
will prioritize the recruitment of military personnel, and secondarily recruit from civilian 
populations as required to fulfill our sample size goal.  
 
When recruiting military personnel to participate in this research, we will conform to 
additional safeguards for the protection of human subjects outlined in DoD Instruction 
(DoDI) 3216.02. These include additional safeguards to protect participants who may be 
more vulnerable to coercion or undue influence.  
 
 
7.0 
Investigational Medical Devices: 
 
For the CES sessions, we will be using the Alpha-Stim AID cranial electrotherapy 
stimulator (CES) medical device, which is an FDA cleared, handheld medical device 
used for the treatment of anxiety, insomnia, and depression (i.e., AID; see Appendix D).  
 
Because our study is using the device with healthy participants, the device is being used 
off-label in a manner that is for investigational use only. As detailed in the Informed 
Consent Form, this means that the device is being used in a study examining its safety 
or effectiveness, and not for the treatment, diagnosis, or prevention of any disorder.  
 
Our intent is to test the effectiveness of the device; its safety has been documented in 
over 100 independent clinical research studies. 
 
8.0 
Incomplete Disclosure or Deception: 
 
We will not use incomplete disclosure or deception. Participants will be informed that 
they will be randomly assigned to a Sham or Active condition. 
 
9.0 
Recruitment Methods: 
 
Participants will be recruited through three mechanisms: (1) Recruitment of Soldier 
Center Human Research Volunteers (HRVs), (2) Recruitment of Active Duty Units 
utilizing the United States Army Forces Command (FORSCOM) troop request process, 
and (3) Recruitment of Civilians. These are detailed below: 
 
1. Recruitment of Soldier Center Human Research Volunteers (HRVs): Participants 
will be recruited from Soldiers in the Human Research Volunteer (HRV) program 

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assigned to Headquarters Research and Development Detachment (HRDD) at 
DEVCOM SC. Personnel from the Human Research Volunteer Program Office 
(HRVPO) will schedule a recruitment briefing about the study for potential HRV 
participants. Superiors of the potential HRV participants (e.g., unit officers, senior 
NCOs, and equivalent civilians) in the chain of command shall not be present at 
the recruitment briefing in which members of units under their command are 
afforded the opportunity to participate as human subjects. This briefing will be 
conducted in a group setting of potential HRV participants. The briefing will be 
completed either in-person or remotely (with the PI or AI briefing remotely or via 
a recorded slideshow, see PowerPoint notes for script). 
 
A representative of the HRVPO may be present during this briefing. Potential 
participants will be asked to participate in the study after being informed of the 
purpose of the study, the nature of the test conditions, the risks associated with 
the study, all procedures affecting a participant’s well-being, a participant’s right 
to discontinue participation at any time without penalty, and that there is no 
penalty for deciding not to participate in the study. 
 
Potential participants will have the opportunity to ask questions and will receive 
copies of the Informed Consent Form to review. After the briefing, potential 
participants will be given time to determine whether or not they wish to 
participate. Subsequent to the briefing, the HRVPO will coordinate with the 
Principal Investigator (PI) to obtain any additional information about the study 
requested by the potential participants. Those individuals interested in 
participating, will communicate this to the HRVPO so a consent meeting with the 
Pl or appropriate research team member can be scheduled. 
 
2. Recruitment of Active Duty Units: Participants will be recruited from active duty 
units utilizing the United States Army Forces Command troop request process or 
other appropriate request processes for troop support.  Principal Investigators (or 
Associate Investigators) will provide information about the study to support these 
requests. The Unit Commander’s support for Soldiers being recruited to 
voluntarily participate in research will be documented in a letter of support.  The 
PI will obtain this letter prior to the study briefing, maintain it as part of study 
records, and send to the DEVCOM SC Human Research Protection Program 
Office. 
 
Participants from active duty units who agree to support the study will be briefed 
on the purpose of the study, the nature of the test conditions, the risks associated 
with the study, all procedures affecting a potential participant’s well-being and 
fitness for duty, a potential participant’s right to discontinue participation at any 
time without penalty, and that there is no penalty for deciding not to participate in 
the study. Superiors of the potential participants (e.g., unit officers, senior non-
commissioned officers (NCOs), and equivalent civilians) in the chain of command 
shall not be present at the recruitment briefing in which members of units under 
their command are afforded the opportunity to consider participation as human 
subjects. The briefing will be completed either in-person (at Soldiers’ home 
stations) or remotely (with the PI or AI briefing remotely or via a recorded 
slideshow, see PowerPoint notes for script). 
 
Potential participants will be asked to participate in the study after being informed 

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of the purpose of the study, the nature of the test conditions, the risks associated 
with the study, all procedures affecting a participant’s well-being, a participant’s 
right to discontinue participation at any time without penalty, and that there is no 
penalty for deciding not to participate in the study. 
 
Potential participants will have the opportunity to ask questions and will receive 
copies of the Informed Consent Form to review. After the briefing, potential 
participants will be given time to determine whether they wish to participate. 
 
3. Recruitment of Civilians: Civilians will be recruited from the community through 
websites advertising research [e.g., Tufts Paid SONA (i.e., not linked to course 
credit)], see Appendix B for recruitment language. This study has no ties to 
classroom grades and/or performance. Potential participants will be informed that 
there is no penalty for deciding not to participate. Interested participants will 
contact an associate investigator listed in the recruitment material to schedule a 
consent discussion.  
 
Participants who agree to learn more about the study will be informed about the 
purpose of the study, the nature of the test conditions, the risks associated with 
the study, all procedures affecting a participant’s well-being, a participant’s right 
to discontinue participation at any time without penalty, and that there is no 
penalty for deciding not to participate in the study.  
 
Potential participants will have the opportunity to ask questions and if they are 
interested in participating, they will sign the Informed Consent Form. Participants 
may directly contact the Principal Investigator or Associate Investigators for 
additional information at any time after the consent discussion. A signed copy of 
the Informed Consent Form will be provided to the participant following the 
consent discussion. 
 
10.0 Consent Process:  
 
Recruitment is detailed above in the Sample Recruitment section. Herein we will 
describe the next phase: consenting. 
 
1. Consent of Soldier Center Human Research Volunteers (HRVs): For the HRV 
participants that expressed interest in participating in the study, a representative 
from the HRVPO will schedule an informed consent meeting between the 
potential HRV participants and the Principal Investigator or a designated 
research team member(s) responsible for consenting participants.  The Principal 
Investigator or study team member consenting the HRV participants will meet 
with them to review the consent document and answer any questions the 
participant may have. The HRV participants will have an opportunity to meet 
individually with the study team member if they would like to discuss or ask 
questions about the study. Those individuals who agree to participate in the 
study will express their understanding by signing an Informed Consent Form. The 
study team member obtaining the consent will also sign the form. The study team 
member obtaining the consent will retain a signed Informed Consent Form and a 
signed copy of the Informed Consent Form will be provided to the participant. A 
representative from the HRVPO may also be present during consenting although 

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their presence is not required for consenting. Consenting will take place at 
DEVCOM SC; the consent form includes screening questions. Participant IDs are 
assigned upon beginning study participation and are never included in email 
communication. The PI and associate investigators communicating via email 
safeguard this information by storing all in a designated folder within their email 
inbox. 
  
2. Consent of (non-HRV) Active Duty Units: For the active duty participants from 
FORSCOM, that expressed interest in participating in the study, an informed 
consent meeting will be scheduled between the potential active duty participants 
and the Principal Investigator or a designated research team member(s) 
responsible for consenting participants. The Principal Investigator or study team 
member consenting the participants will meet with them to review the Informed 
Consent Form and answer any questions the participant may have. The active 
duty participants will have an opportunity to meet individually with the study team 
member if they would like to discuss or ask questions about the study. Those 
individuals who agree to participate in the study will express their understanding 
by signing an Informed Consent Form. The study team member obtaining the 
consent will retain a signed Informed Consent Form and a signed copy of the 
Informed Consent Form will be provided to the participant. Consenting will take 
place at the participants’ home unit. Participant IDs are assigned upon beginning 
study participation and are never included in email communication. The PI and 
associate investigators communicating via email safeguard this information by 
storing all in a designated folder within their email inbox. The consent form 
includes screening questions; consenting will take place either at Tufts 
University, the DEVCOM Soldier Center, or at the units’ home station. 
Participants receive a study in-brief when they travel to participate, explaining the 
study once more.  
 
3. Consent of Civilians. For civilians who respond to the research advertisements 
(in the form of website postings and emails), the Principal Investigator, associate 
investigators, or research assistants will schedule the informed consent interview 
on proposed research and be present at the interview. The PI will distribute the 
Informed Consent Form for the study so that the attendees have the form in hand 
at the time of the interview. At the interview, the PI will provide a briefing to the 
potential participants, using the approved briefing slides. After the briefing, 
potential participants will have the opportunity to ask questions. Those individuals 
who agree to participate in the study will express their understanding by signing 
an Informed Consent Form. The study team member obtaining the consent will 
retain the original completed Informed Consent Form, and a copy will be offered 
to the participant. 
 
11.0 Compensation: 
 
Military personnel who choose to participate in this study will not be compensated. 
Civilians who choose to participate in this study will be compensated at a rate of 
$20/hour. 
 
12.0 Economic Burden: 
 

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There is no economic burden imposed by this study. 
 
13.0 Recording with Audio, Video, or Photographs 
 
This study may optionally involve recording video and/or taking photographs of 
participants performing tasks. If participants agree (on the Informed Consent Form) to 
have these collected, digital videos and/or photographs would be used for educational, 
reporting, or illustration purposes and will be stored for at least 3 years after closure of 
the protocol. The Principal Investigator (PI) will store these digital media files on their 
password-protected computer, and no other study team members will have access. 
 
14.0 Potential Benefits to Participants:  
 
There are no direct benefits to the participants participating in this research. The results 
may help better inform future research on how CES may influence sympathetic nervous 
system responding under conditions of stress. 
 
15.0 Risks to Participants: 
We anticipate three primary risks: 
 
1. Cutaneous Irritation due to StressX Belt: Mild electrical shock to the torso is 
commonly used as an effective, safe method of inducing acute stress in a 
laboratory context. The StressX Pro Belt meets strict safety regulation 
standards set forth by the International Electro Technical Commission 
(document IEC 479-2:1987). Specifically, an excess of 5000mJ of transient or 
capacitive discharge is required to produce direct serious health risk and the 
electrical stimulus system in the study (SETCAN StressX Pro Belt) has a 
maximum output of 92mJ (< 1mA over 150ms), more than 98% lower than 
the threshold to cause health risk (IEC 479-2: 1987). The shocks we deliver 
will be unpleasant but not painful, set to intensity level 3 (of 5) which delivers 
shock for only 60-80ms. In our recently completed study (DEVCOM AC IRB 
protocol 18-007), we completed over 100 sessions using this shock belt, with 
over 1,000 shocks delivered on (self-selected) intensity levels 1 through 3. 
With these settings, only one participant reported mild skin irritation, which 
the AC IRB determined did not qualify as an UPIRTSO. In the present study, 
we will use a fixed shock intensity of 3; while low probability (we estimate 1-
2% incidence given our prior study), if at any time a participant can no longer 
tolerate the shock (either psychologically, or they experience anything more 
intense than mild skin irritation), we will reschedule their session. See Study 
Withdrawal Guidelines (Section 3.0) for details. Participants will be informed 
of shock belt-related risk, and that they can opt-out at any time, in the 
Informed Consent Form. 
 
2. Side Effects due to CES Administration: Cranial electrotherapy stimulation 
(CES) has very low incidence of side effects; when they do occur, they tend 
to include transient dizziness, vertigo, headache, nausea, tinnitus, 
lightheadedness, or skin irritation under the electrodes. The manufacturer of 
the Alpha-Stim AID (EPII) suggests that side effects are seen in less than 1% 
of people using the device, and they are “all mild and self-limiting.” To gain a 

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more objective assessment, we reviewed some CES studies that were not 
associated with (i.e., funded by, conducted by) the manufacturer. In one of 
the largest CES studies to date, none of the 115 participants (N = 60 in Active 
CES group) reported any side effects with six weeks of daily (one hour per 
day; 0.5Hz at 100µA) CES administration (Barclay & Barclay, 2014). In 
another similarly designed study using CES at 0.5Hz and 300µA, 3 of the 12 
participants dropped out of the study due to mild side effects (2 with 
dizziness, 1 with headache) (Bystritsky et al., 2008). In another study, 
participants receiving Active CES treatment for 2 weeks (20 minutes per day; 
N = 7; 2mA at mixed frequencies) showed similarly low rates of reported side 
effects as the Sham CES group (McClure et al., 2015). In our own recent 
study (0.5Hz, 100µA), reports of side effects were also no more frequent in 
the Active versus Sham CES group (Brunye et al., 2022). Given these 
results, we estimate that the rate of side effects with our proposed design will 
be very low (i.e., less than 25%), especially given our individualized daily 
thresholding procedure. See Study Withdrawal Guidelines (Section 3.0) for 
details. Participants will be informed of the risk of CES side effects, and that 
they can opt-out at any time due to discomfort with CES administration, in the 
Informed Consent Form. 
 
3. Psychological distress due to rifle marksmanship: During the baseline and 
follow-up sessions, participants will use a simulated rifle to shoot targets in 
virtual reality. This is like a first-person shooter video game, which research 
shows can variably activate positive (i.e., reward; Koepp et al., 1998) or 
negative (i.e., sadness; Ravaja et al., 2008) emotions. There are vast inter-
individual differences in the emotions experienced during or after video game 
play (Avila et al., 2008; Ravaja et al., 2008), making it difficult to predict which 
individuals will experience positively versus negatively valenced emotion. 
While we believe the risk of psychological distress due to this experience is 
low, it is possible that participants will experience distress during or after the 
rifle marksmanship task (such as sadness, remorse, fear, or anxiety). It is 
unknown whether the rate of distress will be higher or lower in civilians versus 
military personnel; it is also unknown whether the rate of distress will be 
higher or lower in participants with prior video game experience (i.e., 
evidence for desensitization in this manner is equivocal; Regenbogen et al., 
2010). Most importantly, if any participants report negative emotion to the 
research team, we will refer Soldier participants to the Office of Medical 
Support and Oversight (OMSO) at the DEVCOM Soldier Center, refer Tufts 
University student participants to the Tufts University Heath Service, and 
recommend other participants (non-Soldier, non-Tufts affiliated) to speak with 
a qualified mental health practitioner. 
 
We have no reason to believe that the acute stress response will differ between military 
personnel and civilian participants. In fact, research suggests that military personnel do 
not necessarily show higher levels of resilience to stress than civilians (Sohail & Ahmad, 
2021). Furthermore, the primary population of military personnel recruited for this study 
(human research volunteers) has very limited military experience; furthermore, many will 
have a military occupational specialty (MOS) that is not infantry (e.g., engineer, aviation, 
transportation, maintenance), and most (if not all) will have never been deployed outside 
of the US or experienced combat. For these reasons, we do not have any compelling 
reason to believe they will have higher resilience to acute stress relative to civilians.  

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We do not anticipate any issues with virtual reality sickness/simulator sickness given that 
there is no virtual movement or optic flow through the virtual environment. Rather, 
participants are stationary in the environment; indeed, in our recently completed 
protocol, we experienced no reports (in over 200 sessions) of any typical symptoms of 
simulator sickness (e.g., nausea, headache). 
 
If at any time, a civilian participant feels that they have been injured by the shock belt or 
CES and needs emergency care, Emergency Medical Care will be provided by calling 
911, consistent with the Tufts University emergency response. It cannot be determined 
in advance which hospital or clinic will provide care. The participant and/or their 
insurance will be responsible for medical expenses.  
 
If at any time, a Soldier (i.e., DOD healthcare beneficiary) participant feels that they 
have been injured by the shock belt or CES and needs emergency care, Emergency 
Medical Care will be provided by calling 911, consistent with the Tufts University and 
DEVCOM Soldier Center emergency response. This care includes but is not limited to 
free medical care at Army hospitals or clinics. 
 
16.0 Withdrawal of Participants: 
As in all human use protocols, participants will be fully briefed on the purpose, risks and 
procedures of the study before being asked to participate; they will also be informed of 
their right to withdraw from the study at any time without prejudice. During all sessions, 
participants will be allowed to stop participation at any time if they experience any side 
effects (or for any other reason). 
 
As detailed in Section 3.0, we will also withdraw participants from the study based on 
several criteria (see Appendix E for flowchart). All data collected up to the point of 
withdrawal will be destroyed and not analyzed. 
 
17.0 Data Management and Confidentiality: 
 
All data and personal information obtained during this protocol will be considered 
privileged and held in confidence. Each participant will be assigned a unique subject ID 
number. This subject ID number will not contain any personal identifiers such as: name, 
social security number, address, date of birth, zip code, etc. This subject ID number will 
be the only identifier on all data collection instruments (i.e. questionnaires, collection 
forms, data files, and computer records), and used during data collection and analysis. 
Only the study investigators will have access to the Informed Consent Forms containing 
participant names and signatures. A master list linking the participants’ personal 
identifiers with the study participant ID number (i.e., a link) will be kept as an electronic 
document on a password-protected government computer. 
 
Only the PI will have access to the master file (link). Associate investigators and study 
staff will have access only to the coded data and not to personally identifiable 
information. The document that links participants’ names to their ID number will be kept 
for three years after closure following verification and validation of the data, when it will 
be destroyed. The Informed Consent Forms will be destroyed three years after the study 
is closed. When the results of this protocol are published or disseminated in any manner, 

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no information will be included that could reveal identity. Complete confidentiality cannot 
be promised to military participants because information bearing on the military 
participants' health may be required to be reported to appropriate medical or command 
authorities. 
 
Data will be backed-up after each session and inspected by a member of the research 
team. All data and personal information obtained during this protocol will be considered 
privileged and held in confidence. Each participant will be assigned a unique subject ID 
number. This subject ID number will not contain any personal identifiers and will be the 
only identifier on all data collection instruments (i.e. questionnaires, collection forms, and 
computer records). Furthermore, when the results of this protocol are published or 
disseminated in any manner, no information will be included that could reveal identity.  
 
Participants will complete all questionnaires via Qualtrics, which is GDPR (General Data 
Protection Regulation) compliant, and/or tablet computers running a local software 
package (e.g., PsychoPy, SuperLab, or custom C# software). All data will be coded, with 
the only identifier being the participant’s participant ID number assigned at the beginning 
of the study. Access to Qualtrics data is secure and individual permissions set prior to 
the study with access privileges. Once data have been processed, organized, and 
provided to the researchers from both Qualtrics and tablet interfaces, all electronic 
locally stored data will be deleted. 
 
The Informed Consent Forms and any paper copies of the collected data will be stored 
in a locked file cabinet in the Principal Investigator’s office (DEVCOM SC, Building 45, 
L002) for at least 3 years after closure of the protocol. Data will be transferred via DOD 
SAFE, OneDrive, and/or locally approved electronic data transfer sites or cloud 
applications to password-protected computers or held on secure, password-protected 
servers and hard drives for subsequent data analysis. Once the protocol is complete, 
data will be verified and archived to a storage medium (e.g. approved data repository or 
encrypted hard drive) or in the case of hard copies stored in locked file cabinets at the 
Soldier Center for at least 3 years after closure of the protocol.  This data will not contain 
identifying information.  
 
 
18.0 Provisions to Protect the Privacy and Confidentiality of Participants 
and the Research Data: 
 
Participants will be informed of the possibility of future use for the data that is collected 
as part of the consenting process. Data files may also be shared with other government 
and non-government collaborators and contractors for future use.  
 
The data collected in the current study will be used in future studies. There is a 
statement in the Informed Consent Form to obtain consent for future use of the data 
collected. Consent to future use of all data collected will be required for participation in 
the current study and is included under the inclusion criteria.  
 
Participants in this study will consent to allowing their samples to be saved for future 
research that may or may not be directly related to the hypotheses set forth in this 
protocol.  Therefore, once the protocol is closed, samples will be retained for future 
analyses that may or may not align with the aims set forth in the present protocol.  In 
addition, the data may be used as inputs for developing models to assess measures of 

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performance, readiness, recovery, and/or lethality at the individual, small unit and 
platoon-level. DEVCOM Soldier Center or other approved entity may employ a machine 
learning framework within the database to identify groups of individuals who show an 
outcome of interest, and untargeted analysis will search for a set of dependent 
measures that are sensitive to conditional changes (i.e., with versus without an 
environmental stressor). In addition, machine learning algorithms may be used to down-
select dependent measures that correspond to other classification standards. 
 
In all cases, future use of data collected as part of this protocol will be restricted to 
deidentified data after the master linking document (linking participant identity to 
participant ID) has been destroyed. 
 
19.0 Provisions to Monitor the Data to Ensure the Safety of Subjects: 
The PI will periodically inspect the data, especially the CES side effect questionnaire 
responses, to ensure that research staff are following all mitigation procedures outlined 
in this protocol. 
 
20.0 Compensation for Research-Related Injury: 
 
There will be no financial compensation offered for research-related injury.  
 
21.0 Data Sharing and Specimen Banking: 
 
See Section 18.0. 
 
22.0 International Research: 
 
N/A 
 
23.0 Multiple sites:  
This study is a collaboration between the Center for Applied Brain and Cognitive 
Sciences (CABCS) at Tufts University (Medford, MA) and the DEVCOM Soldier Center 
(Natick, MA). Following initial and continuing Tufts University IRB approvals, the 
research team will electronically transmit all study protocol documents to the DEVCOM 
Soldier Center HRPP office and seek secondary Human Research Protections Office 
(HPRO) approvals prior to commencing participant recruitment or any other study 
procedures. Any modifications required by the Soldier Center HRPO to secure 
concurrence will be submitted in an amendment to the Tufts University IRB. The PI will 
be responsible for sharing (via email) the most recent protocol, approvals, and consent 
documentation. Any modifications will be done through the Tufts University IRB and be 
communicated to the Soldier Center HRPO, in accordance with institutional agreements. 
The PI will closely monitor research staff progress at both data collection sites. 
 
24.0 Reliance Agreements/Single IRB: 
 
The Tufts University IRB will serve as the IRB of record. 
 
25.0 Qualifications to Conduct Research and Resources Available: 

Tufts University SBER IRB 
Page 24 of 24 
Revised: November 11, 2018 
Because we recently completed data collection on a very similar study (DEVCOM 
Armament Center IRB #18-007), research assistants have been trained by project 
investigators on the execution of all tasks. Furthermore, investigators attended a virtual 
training seminar offered by the manufacturer of the Alpha-Stim AID device (EPII), which 
included practical and safety-related topics for the effective use of the device. 
 
All study personnel have completed CITI training required for social and behavioral 
research. The PI, Dr. Okano is a Cognitive Neuroscientist and Principal Investigator at 
the Tufts Center for Applied Brain and Cognitive Sciences, and has over 10 years of 
research experience planning, conducting, and managing research in the cognitive 
sciences. She holds a PhD from Tufts University in Cognitive Neuroscience and 
completed post-doctoral training at Massachusetts Institute of Technology (MIT). At 
CABCS, she successfully manages the execution of large-scale research projects 
involving data collection from neurophysiological and physical sensors and has 
substantial experience applying advanced analytical approaches to multimodal data 
streams.