The Combined Effects of Prolonged Sitting and Mental Stress on Vascular and Cerebrovascular Function in Middle-Aged Adults
Phase: NA
Conditions: Sedentary Behavior, Cardiovascular Risk Factor, Mental Stress, Cognitive Change, Cerebrovascular Insufficiency
Sponsor: University of North Carolina, Chapel Hill

 
The Combined Effects of Prolonged Sitting and Mental Stress on 
Vascular and Cerebrovascular Function in Middle-Aged Adults 
 
 
NCT number NCT04207333 
Document Date 12/19/2019 
 

Page 1 of 45 
MASTER PROTOCOL 
 
The Combined Effects of Prolonged Sitting and Mental Stress on Vascular and 
Cerebrovascular Function in Middle-Aged Adults 
 
 
Principal Investigator 
 
Jade Blackwell 
Department of Exercise & Sport Science 
306 Woollen Gym, 
UNC-CH 
Chapel Hill, NC 27599 
 
Co-Investigator(s) 
Jake Diana 
Dr. Lee Stoner 
 
Funding Source: None 
 
Version Date:  12/19/2019 
 
 
 

Page 2 of 45 
PROTOCOL SYNOPSIS 
Study Title:  
The Combined Effects of Prolonged Sitting and Mental Stress on 
Vascular and Cerebrovascular Function in Middle-Aged Adults 
Funding 
UNC Department of Exercise & Sport Science 
Clinical Phase 
Pilot Testing 
Study Rationale 
Prolonged sitting may pose a public health risk through its effects on 
the cardiovascular system, and may lead to impaired whole-body 
cardiovascular health, which includes both vascular and 
cerebrovascular function. These effects may interact with other 
environmental variables, such as stress. However, no study has 
investigated the combined effect of a mental stressor and prolonged 
sitting on vascular and cerebrovascular function. The combined 
effect of prolonged sitting and mental stress may lead to an 
exacerbated effect on vascular, cerebrovascular, and executive 
function. We hypothesize that mental stress with the addition of 
prolonged sitting [SMS] will result in a greater increase in 
peripheral, central and cerebral arterial stiffness and elicit a decrease 
in cerebral perfusion, total blood flow to the brain, middle cerebral 
artery velocity and executive function, compared to mental stress 
without prolonged sitting [CON]. The findings from this study may 
result in a public health message regarding sedentary behavior and 
stress, and will help elucidate the mechanisms behind acute vascular, 
cerebrovascular, and cognitive dysfunction during prolonged sitting. 
Study Objective(s) 
To examine the cardiovascular response to mental stress with or 
without previous exposure to a bout of prolonged sitting.  
Study Design 
Randomized Crossover Design, with two experimental conditions: 
without prolonged sitting + stress [CON] and with prolonged sitting 
+ stress [SMS]. 
Participant  
Inclusion and Exclusion 
Criteria: 
Inclusion 
• Male or female, aged 35 to 59 years 
Exclusion 
• Any known cardio-metabolic disorders 
• Taking medications known to affect cardiovascular function 
• Report cigarette smoking 
• Pregnant women 
Number of Participants  
20 
Study Duration 
1 year 
Study Phases 
 
Familiarization: to all devices 
CON: mental stress without prolonged sitting 
SMS: mental stress with prolonged sitting 
Design 
Multi-visit randomized crossover trial 

Page 3 of 45 
Primary & Secondary 
Outcomes 
Cerebrovascular 
Primary: Brain PWV 
Secondary: Executive Function & Neurovascular Coupling 
Vascular 
Primary: Brachial-Femoral PWV 
Secondary: Carotid-femoral PWV, femoral-ankle PWV, 
augmentation index, central blood pressures (cSBP, cDBP) 
Descriptive/ Mechanistic 
Outcomes 
Cerebrovascular: 
Cerebral Perfusion 
Middle Cerebral Artery Velocity 
Total Brain Blood Flow 
Static Cerebral Autoregulation 
 
Vascular: 
Peripheral blood pressure 
Heart rate 
Baroreflex Sensitivity 
Heart Rate Variability 
Total hemoglobin  
Tissue saturation index 
Accelerometer 
Statistical and Analytic 
Plan 
All statistical procedures will be competed with SPSS Statistics 
version 25.0 (SPSS, IN., Durham, NC, USA) and Jamovi v0.9 
(Jamovi, UK). The α-level will be set a prior at 0.05 for all statistical 
procedures. Descriptive statistics and mechanistic outcomes will be 
collected to compare similarities amongst the sample group and to 
eliminate the potential for extraneous covariates. Answering the 
research question will require a linear, multi-level mixed model. 
Data and Safety 
Monitoring Plan 
No participants will be identified in any report or publication of this 
study. Participants will be identified via identification code for data 
collection purposes. All electronically collected data will be stored 
on a computer that only team members Onyen password will allow 
access to study information. This computer will be located in the 
Graduate Student Office of the Applied Physiology Laboratory at the 
University of North Carolina at Chapel Hill. Data analysis 
procedures will be performed on secure computers within Fetzer 
Hall, with which password is required to access. 
 
 
 

Page 4 of 45 
CONTENTS 
PROTOCOL SYNOPSIS................................................................................................................ 2 
ABBREVIATIONS ........................................................................................................................ 7 
1  
BACKGROUND AND RATIONALE ................................................................................... 9 
Introduction and Rationale .......................................................................................................... 9 
1.1  
Objective(s) ..................................................................................................................... 9 
1.2  
Aims ................................................................................................................................ 9 
2  
STUDY PROCEDURES ...................................................................................................... 10 
2.1  
Participants .................................................................................................................... 10 
Inclusion Criteria .................................................................................................................. 10 
Exclusion Criteria ................................................................................................................. 10 
2.2  
Allocation to Treatment Groups and Blinding.............................................................. 10 
2.3  
Experimental Design ..................................................................................................... 10 
Study Design ......................................................................................................................... 10 
Overview ............................................................................................................................... 10 
Experimental Set-Up ............................................................................................................. 12 
Familiarization ...................................................................................................................... 12 
Experimental Visits ............................................................................................................... 13 
2.4 Experimental Procedures .................................................................................................... 14 
Protocol 1: Mental Arithmetic Test ...................................................................................... 15 
2.5  
Measurements ............................................................................................................... 16 
DEVICE 1: VICORDER® ................................................................................................... 17 
DEVICE 2: Transcranial Doppler ......................................................................................... 17 
DEVICE 3: Non-Invasive Blood Pressure (NIBP) ............................................................... 17 
DEVICE 4: Doppler Ultrasound ........................................................................................... 17 
DEVICE 5: Continuous Wave Near-Infrared Spectroscopy with Spatially Resolved 
Spectroscopy (cwNIRS-SRS or NIRS) ................................................................................. 17 
DEVICE 6: ActiGraph .......................................................................................................... 18 
DEVICES 7 & 8: Data Acquisition (PowerLab) and Data Processing (LabChart) .............. 18 
Cognitive Measurement 1: Trails A+B Tests ....................................................................... 18 
Cognitive Measurement 2: Verbal Fluency Task ................................................................. 19 
Cognitive Measurement 3: Memory Recall .......................................................................... 19 
Cognitive Measurement 4: Brain Fog ................................................................................... 19 
Data Reduction...................................................................................................................... 19 

Page 5 of 45 
2.6 Timeline .............................................................................................................................. 20 
Study Time Commitment ...................................................................................................... 20 
Familiarization Session ......................................................................................................... 21 
CON Experimental Session .................................................................................................. 21 
SMS Experimental Session ................................................................................................... 22 
3  
MEASUREMENT DEVICES .............................................................................................. 23 
Device 1: VICORDER® ........................................................................................................... 23 
Device 2: Transcranial Doppler (TCD) .................................................................................... 24 
Device 3: Non-Invasive Blood Pressure (NIBP) ...................................................................... 26 
Device 4: Doppler Ultrasound .................................................................................................. 27 
Device 5: Continuous Wave Near-Infrared Spectroscopy with Spatially Resolved Spectroscopy 
(cwNIRS-SRS or NIRS) ........................................................................................................... 29 
Device 6: ActiGraph ................................................................................................................. 31 
DEVICES 7 & 8: Data Acquisition (PowerLab) and Data Processing (LabChart) .................. 32 
4  
STATISTICAL CONSIDERATIONS.................................................................................. 34 
4.1  
Study Outcomes ............................................................................................................ 34 
4.2  
Statistical Analysis ........................................................................................................ 34 
4.3  
Tabulation of Results .................................................................................................... 35 
4.4  
Missing Values.............................................................................................................. 35 
4.5 Quality Control ................................................................................................................... 36 
4.6  
Sample Size ................................................................................................................... 36 
4.7  
Statistical Support ......................................................................................................... 36 
5  
Participant Screening, Withdrawal & Completion5 n failure procedures ............................ 37 
5.2  
Participant withdrawal .................................................................................................. 37 
5.3  
Participant Completion ................................................................................................. 37 
6  
SAFETY MONITORING & MANAGEMENT ................................................................... 38 
6.1  
Adverse Event Risk....................................................................................................... 38 
6.2 Responding Adverse Events ........................................................................................... 39 
6.3 Confidentiality ................................................................................................................ 39 
6.4 Participant Withdrawal ................................................................................................... 39 
7 
DATA COLLECTION AND MANAGEMENT .................................................................. 40 
7.1  
Quality Assurance ......................................................................................................... 40 
7.2  
Database Security .......................................................................................................... 40 
7.3  
Confidentiality .............................................................................................................. 40 
7.4  
PHI Security .................................................................................................................. 40 

Page 6 of 45 
7.5  
Risk of Deductive Disclosure ....................................................................................... 41 
7.6  
Data Collection ............................................................................................................. 41 
7.7 
Data Entry ..................................................................................................................... 41 
7.8  
Data Editing .................................................................................................................. 41 
7.9  
Database Documentation .............................................................................................. 41 
7.10  
Pilot Testing of Operations ....................................................................................... 41 
8 
RECRUITMENT STRATEGYRECRUITMENT STRATEGY .......................................... 42 
8.1 
Recruitment Strategies .................................................................................................. 42 
8.2  
Recruitment Personnel .................................................................................................. 42 
8.3  
Protection of Privacy..................................................................................................... 42 
8.4 
Contacting Participants ................................................................................................. 42 
8.4 
Efforts to ensure equal access to participation among women and minorities ............. 42 
9 
STUDY MANAGEMENT/ CONSENTSTUDY MANAGEMENT/ CONSENT ............... 43 
9.1 Consent and Institutional Review Board (IRB) Approval .................................................. 43 
9.2 
Efforts to Minimize Influencing the Participant’s Decision to Participate ................... 43 
9.2 Required Documentation .................................................................................................... 43 
9.3 Adherence to the Protocol ................................................................................................... 43 
9.4 Emergency Modifications ................................................................................................... 43 
10 REFERENCESREFERENCES ............................................................................................ 44 
 
 
 

Page 7 of 45 
   
ABBREVIATIONS 
 
ABBREVIATION 
DEFINITION 
ADRD 
Alzheimer’s Disease and Related Dementia 
AIx 
Augmentation Index 
APL 
Applied Physiology Laboratory 
BF 
Blood Flow 
bfPWV 
Brachial-Femoral Pulse Wave Velocity 
BP 
Blood Pressure 
CBP 
Central Blood Pressure 
cfPWV 
Carotid-Femoral Pulse Wave Velocity 
cm 
Centimeters 
CON 
Control Experimental Session 
CONSORT 
Consolidated Standards of Reporting Trials 
CV 
Cardiovascular 
CVD 
Cardiovascular Disease 
CVH 
Cardiovascular Health 
CVS 
Cardiovascular System 
cwNIRS-SRS or NIRS 
Continuous Wave Near-Infrared Spectroscopy 
DBP 
Diastolic Blood Pressure 
ECG  
Electrocardiogram 
EF 
Executive Function 
faPWV 
Femoral-Ankle Pulse Wave Velocity 
HIPAA 
Health Insurance Portability and Accountability Act 
HR 
Heart Rate 
hr 
Hours 
HRV 
Heart Rate Variability 
ID 
Identification Number 
IRB 
Institutional Review Board 
kg 
Kilograms 
L 
Straight-line length or distance 
m/s 
Meters per second 
MAT 
Mental Arithmetic Test 
MCA 
Middle Cerebral Artery 
MCAv 
Middle Cerebral Artery Velocity 
min 
Minutes 
mmHg 
Millimeters of Mercury 
MS 
Mental Stress or Mental Stressor  
N/A 
Not Available 
NIBP 
Non-Invasive Blood Pressure 
PA 
Physical Activity 
PCA 
Posterior Cerebral Artery 
PHI 
Personal Health Information 

Page 8 of 45 
PI 
Principal Investigator 
PPG 
Photoplethysmography or Photoplethysmography 
PS 
Prolonged Sitting 
PWA 
Pulse Wave Analysis 
PWV 
Pulse Wave Velocity 
PWV/A 
Pulse Wave Velocity and Analysis 
RDM 
Research Database Management 
SBP 
Systolic Blood Pressure 
SD 
Standard Deviation 
sec or s 
Seconds 
SMS 
Sitting & Mental Stress Experimental Session 
TCD 
Transcranial Doppler 
tHb 
Total Hemoglobin 
TMT 
Trails Mental Tests or Trails A + B 
TSI 
Tissue Saturation Index 
TT or ΔT 
Transit Time 
UNC 
University of North Carolina at Chapel Hill 
US 
Ultrasound 
VFT 
Verbal Fluency Test 
VPN 
Virtual Private Network 
X 
Mean or Average 
 
 

Page 9 of 45 
1  
BACKGROUND AND RATIONALE 
Introduction and Rationale  
Prolonged sitting (PS) has gained attention over the past decade due to the sitting epidemic that 
takes place in the workplace.1,2 Participating in a sedentary lifestyle is common due to 
occupation, technology and vehicle transportation. Sedentary behavior (SB) has been shown to 
be a risk factor for cardiovascular disease (CVD) and all-cause mortality. Additionally, a 
sedentary lifestyle has been linked to chronic cognitive dysfunction, thus playing a role in the 
appearance of Alzheimer’s Disease and Related Dementia’s (ADRD)3. The available research 
demonstrates that chronic SB is associated with a reduction in brain volume4 and a decline in 
cognition, in particular executive function (EF)5,6. Although, there is a level of uncertainty in the 
mechanism(s) connecting repeated acute SB exposure to chronic cerebrovascular complications.  
 
In addition to cerebrovascular implications as a result of sedentarism and stress, central and 
peripheral cardiovascular function and structure are disrupted7–9. In contrast to our understanding 
of cerebrovascular function, the physiological mechanisms in which a mental stressor or a 
sedentary behavior like PS10–12 impact systemic cardiovascular function is better understood. 
Pooling of blood in the lower-limbs is the first cardiovascular disruption as a result of PS that 
eventually leads to failures in regulatory mechanism that maintain healthy function11,13–15. 
Repeated activation of neurohormonal pathways due to mental stress is known to impair 
cardiovascular health16,17. It is less clear how these negative stimuli impact the CVS acutely 
when experienced in combination. Developing our knowledge on the topic of cerebrovascular 
and vascular health is necessary to develop societal and behavioral solutions for the sedentarism 
and stress epidemics that significantly burden our healthcare system18–20 and economic 
productivity21. This study will provide new insights on the physiological mechanisms these 
health behaviors take to negatively change the responses of the CVS.  
 
1.1  
Objective(s) 
To examine the cardiovascular response to mental stress with or without previous exposure to a 
bout of prolonged sitting.  
 
1.2  
Aims 
Two primary aims will be addressed: 
 
1. Explore the effects of mental stress with and without a bout of prolonged sitting on 
cerebrovascular function. 
2. Explore the effects of mental stress with and without a bout of prolonged sitting on 
vascular function. 
 
 
 

Page 10 of 45 
2  
STUDY PROCEDURES  
This study will be reported in accordance with Consolidated Standards of Reporting Trials 
(CONSORT) guidelines22.  
 
2.1  
Participants 
We will recruit 20 adult (35-59 y) male and female participants. A healthy population sample 
will be recruited to mitigate the risk of age- or disease-related influences on the study outcomes. 
 
Inclusion Criteria 
• Male or female participants will be between the ages of 35-59 years old. 
 
Exclusion Criteria 
• 
Any known cardio-metabolic disorders 
• 
Taking medications known to affect cardiovascular function 
• 
Report drug or alcohol abuse 
• 
Report cigarette smoking 
• 
Pregnant women 
 
2.2  
Allocation to Treatment Groups and Blinding  
The randomization procedure will be performed by a research assistant. Participants will be 
randomized into either CON or SMS intervention for the first visit. This will be done during the 
familiarization session using an online randomization software (www.randomizer.org). 
 
2.3  
Experimental Design 
Study Design 
This study is a multi-visit (three in total) randomized crossover trial.  
 
Overview 
 
During a single familiarization session, the research team will obtain informed consent, and will 
describe to participants the purposes and procedures of this study. They will then be exposed to 
all experimental devices on in a quiet, dimly lit and environmentally controlled room. 
Participants will return to the same location for two experimental conditions following these pre-
assessment guidelines: 
• Fasted (> 12 hours), consuming only water. 
• No caffeine consumption 12 hours prior to testing. 
• No vigorous exercise 24 hours prior to testing. 
• No alcohol consumption 24 hours prior to testing. 
 
 
 
 
 
 

Page 11 of 45 
Table 1. Study Variables 
 Type 
Measure 
Independent (Conditions) 
Control (CON): Exposure to MS without PS 
 
Sitting-Mental Stress (SMS): Exposure to MS after period of PS 
Primary Outcomes 
Brain PWV 
 
Brachial-femoral PWV 
Secondary Outcomes 
Executive function 
 
Neurovascular coupling 
 
Carotid-femoral PWV 
 
Augmentation index 
 
Femoral-ankle PWV 
 
 
 

Page 12 of 45 
Experimental Set-Up 
 
 
Figure 1. Experimental setup for to assess full body cardiovascular health. Each experimental 
device responsible for collecting data is shown in this picture and close ups. 
 
Familiarization 
Participants will be familiarized with all experimental procedures and then given the opportunity 
to ask any questions prior to providing written consents. Subjects will be brought to an upright, 
seated position using an Armedica AM353 Hi-lo Treatment Table (Tiger Medical, 
TIGER#TM83695) with feet flat on the ground with approximately 90 degrees of knee flexion. 
If feet cannot touch the ground, a platform will be used. The angle of recline of the chair will be 
measured, as well as the distance from the bottom of the chair to the ground to ensure equivalent 
chair placement for the second visit. 
 
 
 

Page 13 of 45 
Experimental Visits 
Refer to both Table 1 and Figure 1 for a breakdown of when experimental measures will be 
taken through both visits. 
 
Participants will arrive to the Applied Physiology Laboratory (APL) between 6:00 and 10:00 
AM fasted (for Visit 3: 2-5 days following the Visit 2). Participants will be fasted and refrain 
from caffeine intake for at least 12 hours, and alcohol and strenuous physical activity for at least 
24 hours prior to arrival. Upon arrival, height and weight will be recorded followed by 10 
minutes of quiet rest in the supine position (white solid boxes in Figure 1). During these 10 
minutes, the subject will be fitted with a NIRS probe on the prefrontal cortex and medial 
gastrocnemius. The NIBP, TCD, and VICORDER® devices will also be affixed to the 
participant during this time period during this time. Sitting periods (solid gray boxes in Figure 1) 
will begin for both conditions once all devices are attached to the participant, the subject is 
shifted to an upright seated position, and at least 10min of supine rest has been recorded. During 
the CON visit, subjects will undergo a brief 20min sitting period. For the SMS condition, 
subjects will be asked to sit still and quietly for 2 hours while watching a non-stimulating 
documentary. Each condition will receive the MS (black boxes in Figure 1) at the conclusion of 
the sitting periods in both CON and SMS. After exposure to the MS in both conditions, data 
collection procedures for this protocol will be completed  for VICORDER® immediately after 
the MS and every 5 minutes for 25 minutes (See Figure 1 for visual details on how and when 
data is being collected). Brain BF will be assessed by US 20 minutes post-completion of the 
MAT. Finally, a battery of cognitive tests (Verbal Fluency Test and Trails A+B tests) will be 
administered to the participant (boxes with diagonal shades in Figure 1).  
 
 

Page 14 of 45 
2.4 Experimental Procedures 
 
 
 
 
Figure 2. A visualization of the experimental protocol. Maintenance of external validity for this study will require exposure 
of the MS to be time-matched between both experimental conditions. The use of accelerometry will allow investigators to 
observe physical activity and sleep patterns to ensure uniformity in pre-assessment behaviors. All time points are expressed 
relative to the time of completion of the mental stressor. Abbreviations: BF, blood flow; PWV/A, Pulse wave velocity & 
analysis; (1), collection of PWV/A for the primary outcome, bfPWV/A will be collected at this time point only; (3) 
collection of PWV/A for bfPWV, cfPWV, and faPWV; PA, Physical activity; t, time; NIRS, Near-infrared spectroscopy; 
NIBP, Non-invasive blood pressure; TCD, Transcranial Doppler.  

Page 15 of 45 
Protocol 1: Mental Arithmetic Test 
 
Test Structure 
Application 
 
 
 
 
 
• 
Test duration: Approximately 5min 
• 
Participant is in seated upright position 
• 
At the beginning of the test, the researcher 
will call out a four digit number and ask 
the participant to subtract either 7 or 13 
• 
Correct responses will not be recorded as 
an outcome for this study 
• 
A metronome will play at a pre-set tempo. 
Subjects will be instructed to subtract 7 or 
13 for one minute from the original 
number continuously 
• 
Each minute, a new four digit number will 
be called out and the participant must 
subtract the 7 or 13 from the number 
 
 
The MS used for this protocol is the Mental Arithmetic Test (MAT), a commonly used exam to 
impose a small psychological stimulus on the participant. This test will approximately last for 
five minutes. The researcher will call out a four-digit number and ask the participant to subtract 
either seven or 13. Correct responses will not be recorded as an outcome for this study. During 
this test a metronome will play at a pre-set tempo. The subjects will be required to conduct 
continuous subtraction with the tempo to the best of their ability. Every minute, a new four digit 
number will be called out and the participant must subtract the seven or 13 from the number.  
 
 
 
 
 
 
 

Page 16 of 45 
2.5  
Measurements 
 
Table 2. Summarization of measures used for the protocol, “The Combined Effects of Prolonged 
Sitting and Mental Stress on Vascular and Cerebrovascular Function in Middle-Aged Adults”. 
Each experimental variable is listed under the “Outcomes” column with its designation as either a 
primary, secondary, or mechanistic measure listed under the “Role Classification” column. The 
“Device” column names the equipment used to collect the data. Abbreviations: TCD, 
Transcranial Doppler; CON, Control condition; SMS, Sitting mental stress condition; VFT, Verbal 
fluency test; TMT, Trail making test; NIRS, Near-infrared spectroscopy; cwNIRS-SRS, 
Continuous-wave near-infrared spectroscopy. Minute values are expressed relative to the 
administration of the MAT. 
 
 
 

Page 17 of 45 
DEVICE 1: VICORDER®  
PWV is calculated by dividing arterial path length (L) by the pulse transit time (TT) between a 
proximal and distal arterial segment. Measurements for L are acquired by recording the straight 
line distance between the proximal and distal cuff, per manufacturer guidelines. For PWV 
measures including the carotid artery, the straight line distance from the carotid artery to the sternal 
notch is included in calculations for L.  
 
Transit time is calculated by the Vicorder software’s proprietary algorithm that measures the time 
between the foot of the proximal pressure waveform to the foot of the distal pressure waveform. 
Pressure waveforms will be simultaneously captured using volume displacement cuffs. A small 
balloon cuff is placed over the carotid artery. The balloon used to generate the carotid pulse wave 
does not cause discomfort as it inflates to sub-diastolic blood pressure (~50 mmHg). 
 
DEVICE 2: Transcranial Doppler  
Brain PWV and Middle cerebral artery velocity (MCAv) will be measured using continuous 
bilateral TCD (ST3, Spencer Technologies). MCAv accounts for 70-80% of the brain’s total 
perfusion25. The data will be acquired continuously (50 Hz) using an analog-to digital convertor 
(PowerLab 30, ADInstruments). 
 
DEVICE 3: Non-Invasive Blood Pressure (NIBP) 
The PowerLab data acquisition system (PowerLab 30 series, ADInstruments, CO, USA) will be 
used to simultaneously acquire signals from each device and from ECG. A standard Lead II ECG 
will be recorded from three electrodes placed on the right and left shoulders, and just below the 
left rib. The signals from the ECG and two test devices will be sampled a frequency was 1000 Hz, 
providing a temporal resolution of 1ms. 
 
DEVICE 4: Doppler Ultrasound 
A commercial ultrasound device equipped with 11-3 MHz linear array probe will be used. 
Experimenters will assess blood flow in three cerebral arteries: common carotid, internal carotid, 
and vertebral. 
 
DEVICE 5: Continuous Wave Near-Infrared Spectroscopy with Spatially Resolved 
Spectroscopy (cwNIRS-SRS or NIRS) 
A continuous, non-invasive system to measure cerebral perfusion. The near-infrared spectroscopy 
probe used in this protocol emits a single frequency of light (cwNIRS) to monitor relative change 
in total hemoglobin. Using the spatially-resolved spectroscopy (SRS) technique, cwNIRS can 
estimate absolute measures of perfusion (e.g. total hemoglobin, tissue saturation index). The 
cwNIRS-SRS device will be positions on the forehead, approximately 3cm to the right of the 
center, directly over the eyebrow. An additional probe will be placed over the medial 
gastrocnemius to observe venous pooling of blood in the lower-limbs. The location of both probes 
will be marked to ensure identical placement for the next experimental session. The device will be 
covered with a dark opaque cloth to prevent signal contamination by ambient light as per 
manufacturer recommendations. 
 
 

Page 18 of 45 
DEVICE 6: ActiGraph 
An ActiGraph accelerometer will be distributed to the research participant at the conclusion of the 
familiarization session along with a sleep journal packet. The subject will be asked to record on 
the timeline when he/she began to sleep and awaken each day until the conclusion of his/her 
participation in this study. The device will be worn on the individual’s non-dominant wrist 
according to manufacturer guidelines. The data collected from this device will be used for one 
purpose: 
• Validate the subject’s compliance with pre-testing behavioral restrictions for the purpose 
of internal validity: 
o Waking up at similar times on the mornings of both experimental session 
 
DEVICES 7 & 8: Data Acquisition (PowerLab) and Data Processing (LabChart) 
For the TCD and NIBP, devices, data acquisition and processing will be automated using the 
accompanying software.  
 
For the two test devices, the PowerLab data acquisition system (PowerLab 30 series, 
ADInstruments, CO, USA) will be used to simultaneously acquire signals from each device and 
from ECG.  
 
LabChart 8 (ADInstruments) will record and process the data acquired using PowerLab. The ECG 
signal does not require any post-processing, the software filters ensure a distinct R peak. The foot 
of the pressure waveforms from the NIBP and PPG devices will be identified using a standard 
second derivative function. Offline, a custom macro will identify the peak of the R wave and the 
peak of the second derivative of each pressure waveform, then calculate the time between said 
peaks. 
 
Cognitive Measurement 1: Trails A+B Tests 
 
Test Structure & Application 
The Trail-Making Test – Part A, the circles are 
numbered 1 – 25, and the subject will draw 
lines to connect the numbers in ascending 
order. Part B presents numbers and letters 
placed in a semi-random fixed order, in such a 
manner to avoid overlapping lines being 
drawn. The subject will connect 25 encircled 
numbers and letters in numerical and 
alphabetical order, alternating between the 
numbers and letters. For example, the first 
number ‘‘1’’ is followed by the first letter ‘‘A’’ 
followed by the second number ‘‘2’’ then 
second letter ‘‘B’’ and so on23 Results for both 
TMT A and B will be reported in seconds. 
 
 
 
 
 
 
 
 
 
 

Page 19 of 45 
Cognitive Measurement 2: Verbal Fluency Task 
 
Test Structure 
Application 
Tests of verbal fluency evaluate an 
individual’s ability to retrieve specific 
information within restricted search 
parameters24. The two subtests are (1) 
semantic fluency, and (2) phonemic 
fluency. The total number from each 
test will be summed and equate to the 
subjects’ cognitive performance. 
 
 
 
 
Cognitive Measurement 3: Memory Recall 
Participants will be read word lists from the Hopkins Verbal Learning Test at the rate of 1 word 
every 2 seconds pre-test and post- cognitive testing period. The Hopkins Verbal Learning test word 
lists contain 12 words, 4 words from 3 semantic categories. 
 
Cognitive Measurement 4: Brain Fog 
Brain fog will be subjectively measured using a modified Borg Rate of Perceived Exertion (RPE) 
scale as there is no known reliable and valid objective measure of brain fog. This will be 
administered pre-test and post-cognitive testing period. 
 
Data Reduction 
The data collection periods are listed in Table 3. Minute values are noted in Figure 1 and are 
expressed relative to the administration of the MAT. 
 
Table 3. Data reduction methods for experimental protocol 
Device 
Outcomes 
CON (min) 
SMS (min) 
Reduction 
VICORDER
® 
bfPWV 
-5, 0, 5, 10, 
15, 20, 25 
-115, -15, 0, 5, 
10, 15, 20, 25 
Measures done in 
triplicate. Closest 2 
will be averaged for 
mean and variance 
reported 
 
cfPWV 
-5, 25 
-115, -15, 25 
 
faPWV 
-5, 25 
-115, -15, 25 
 
CBPs 
-5, 0, 5, 10, 
15, 20, 25 
-115, -15, 0, 5, 
10, 15, 20, 25 
 
Peripheral BPs 
-5, 0, 5, 10, 
15, 20, 25 
-115, -15, 0, 5, 
10, 15, 20, 25 
 
Augmentation index 
-5, 0, 5, 10, 
15, 20, 25 
-115, -15, 0, 5, 
10, 15, 20, 25 
TCD 
Brain PWV 
Continuous 
Continuous 
30s selections made 
by 
following 
a 
 
MCA velocity 
Continuous 
Continuous 

Page 20 of 45 
 
Static cerebral autoregulation 
Continuous 
Continuous 
prearranged 
protocol. Data for the 
outcome in the time 
epoch 
will 
be 
averaged and SD 
will be reported 
 
Neurovascular coupling 
Continuous 
Continuous 
NIBP 
Baroreflex sensitivity 
Continuous 
Continuous 
30s selections made 
by 
following 
a 
prearranged 
protocol. Data for the 
outcome in the time 
epoch 
will 
be 
averaged and SD 
will be reported 
 
Heart rate variability 
Continuous 
Continuous 
 
Heart rate 
Continuous 
Continuous 
Ultrasound 
Cerebrovascular BF 
-5, 25  
-120, -5, 20 
 
NIRS 
Cerebral perfusion 
Continuous 
Continuous 
30s selections made 
by 
following 
a 
prearranged 
protocol. Data for the 
outcome in the time 
epoch 
will 
be 
averaged and SD 
will be reported 
 
Tissue saturation index 
Continuous 
Continuous 
 
Venous pooling 
Continuous 
Continuous 
ActiGraph 
Physical activity 
Continuous 
Continuous 
 
 
Sleep duration 
Continuous 
Continuous 
Cognitive 
Tests 
Executive Function 
25 
25 
A single score for 
each cognitive test is 
received from each 
participant from each 
experiental visit 
 
 
2.6 Timeline 
 
Study Time Commitment 
 
Stage 
Time (mins) 
Description 
Recruitment 
10 
Via email, flyer and/or class 
presentation 
Familiarization Session 
35 
Familiarize test subject with the 
purpose 
of 
the 
study, 
all 
experimental devices and the 
timeline of each experimental 
condition 
Experimental Visits  
255 
All measurements, single visit 
Total 
300 
 
 
 
 

Page 21 of 45 
 
Familiarization Session 
Time (mins) 
Action 
 
0:00-5:00 
Share adult consent form with participant. Questions and concerns 
will be heard on the protocol and purpose of this study. 
5:00-10:00 
Discuss the underlying physiology and purpose that lead to the 
creation of this study. Discuss how this study will answer the research 
questions. 
10:00-30:00 
The researcher will attach each device to the participant to familiarize 
him/her to the protocol. No data will be collected for later analysis 
during this period. 
30:00-35:00 
Subject and researchers will schedule the date and time for CON and 
SMS. ActiGraph will be attached to the research participant. A 
document will be provided detailing all behavioral restrictions (e.g. 
abstinence from caffeine, alcohol) and instructions on wearing the 
ActiGraph will be provided.  
Session End – Total time spent in APL: 35 minutes 
 
CON Experimental Session 
Time (mins) 
Action 
Measurement Order 
EXAMPLE START 
TIME 
-30:00 - -25:00 
Anthropometrics 
Weight (kg) 
Height (cm) 
BMI (km/m2) 
10:15 AM 
-25:00 
Begin Supine Rest 
N/A 
10:20 AM 
-25:00 - -15:00 
Affix devices to 
participant. 
Path lengths (cm) 
10:20 AM 
-15:00 
End Supine Rest. Begin 
continuous data 
collection from devices. 
Begin sitting period  
TCD measures 
NIRS measures 
NIBP measures 
 
10:30 AM 
-15:00 - -5:00 
Ultrasound data 
collection 
Cerebrovascular blood 
flow (common carotid, 
internal carotid, and 
vertebral arteries) 
10:30 AM 
-15:00 - -5:00 
VICORDER® data 
collection 
bfPWV/A 
cfPWV/A 
faPWV/A 
10:30 AM 
-5:00-0:00 
Mental Arithmetic Test 
N/A 
10:40 AM 
00:00-1:00 
VICORDER® data 
collection 
bfPWV/A immediately 
after MAT 
10:45 AM 
5:00-20:00 
VICORDER® data 
collection 
bfPWV/A every 5m post-
MAT for addition 20m 
10:50 AM 
25:00-30:00 
VICORDER® data 
collection 
All measures of PWV/A 
10m post-MAT 
11:10 AM 
30:00-40:00 
Cognitive tests 
Trails A+B 
VFT 
11:15 AM 
Test End – Total time spent in APL: 75 minutes 

Page 22 of 45 
 
SMS Experimental Session 
Time (mins) 
Action 
Measurement Order 
EXAMPLE 
START TIME 
-140:00 - -135:00 
Anthropometrics 
Weight (kg) 
Height (cm) 
BMI (kg/m2) 
8:25 AM 
-135:00 
Begin Supine Rest 
N/A 
8:30 AM 
-135:00 - -125:00 
Affix devices to 
participant. Collect 
path lengths required 
for measures from 
PWV/A 
Path lengths (cm) 
8:30 AM 
-125:00 
End Supine Rest. 
Begin continuous data 
collection from 
devices. 
Begin prolonged 
sitting period  
TCD measures 
NIRS measures 
NIBP measures 
 
8:40 AM 
 -120:00 -115:00 
Ultrasound data 
collection 
Cerebrovascular blood flow 
(common carotid, internal 
carotid, and vertebral 
arteries) 
8:45 AM 
-115:00 - -110:00 
VICORDER® data 
collection 
bfPWV/A 
cfPWV/A 
faPWV/A 
8:50 AM 
-20:00 - -15:00 
Ultrasound data 
collection 
Cerebrovascular blood flow 
(common carotid, internal 
carotid, and vertebral 
arteries) 
10:25 AM 
-15:00 - -5:00 
VICORDER® data 
collection 
bfPWV/A 
cfPWV/A 
faPWV/A 
10:30 AM 
-5:00 
End prolonged sitting 
period 
N/A 
10:40 AM 
-5:00 – 0:00 
Mental Arithmetic Test 
N/A 
10:40 AM 
00:00-1:00 
VICORDER® data 
collection 
bfPWV/A immediately 
after MAT 
10:45 AM 
5:00-20:00 
VICORDER® data 
collection 
bfPWV/A every 5m post-
MAT for addition 20m 
10:50 AM 
25:00-30:00 
VICORDER® data 
collection 
All measures of PWV/A 
10m post-MAT 
11:10 AM 
30:00-40:00 
Cognitive tests 
Trails A+B 
VFT 
11:15 AM 
Test End – Total time spent in APL: 180 minutes 
 
 
 

Page 23 of 45 
3  
MEASUREMENT DEVICES 
 
Device 1: VICORDER® 
 Device 
 
 
 
 
• Company: SMT Medical 
• Model: VICORDER®: Arterial Stiffness 
Model 
• Location: Wurzburg, Germany 
• Oscillometric cuffs inflate at sub-systolic 
pressures to detect changes in the pressure 
waveform.  
• Different sized cuffs are available. 
 
Example Set-up 
 
 
 
• The cuffs can be placed on any 2 arterial 
segments.  
• In the figure to the left, pressure cuffs placed 
over the brachial and femoral arteries. 
• A small balloon is placed over the carotid 
artery to obtain a measurement for cfPWV. A 
low pressure (50 mmHg) is used for this site 
with minimal discomfort. 
Raw Data 
 
 
 
• Top display = pressure waveform from 
proximal arterial segment 
• Bottom display = pressure waveform from 
distal arterial segment 
• Intrinsic software automatically detects the 
foot of the waveform of each arterial segment  
 
 
 
Data Analysis 
 
• A = Site of proximal artery 
• B = Site of distal artery 
• Pulse wave velocity (PWV) = distance (D) / 
transit time (TT) 
• Straight line distance (L) = Distance 
between two cuffs as measured according to 
manufacturer guidelines 

Page 24 of 45 
 
 
• Transit Time (ΔT or TT) = Time between 
foot of proximal and distal waveforms. 
• Oscillometric pressure waveforms will be 
determined at the brachial artery of the non-
dominant arm using the VICORDER® in 
order to determine brachial and central 
arterial blood pressures. 
Data Reduction 
 
 
• L is plugged into software prior to 
measurement. 
• TT is averaged across 3 waveforms, and used 
to calculate one PWV 
• Process repeated 3 times, for 3 PWVs per 
measurement cycle. 
• Closest 2 PWVs averaged and used as 
outcome for measurement cycle. 
 
 
Device 2: Transcranial Doppler (TCD) 
 Device 
 
 
 
• Company: Compumedics DWL 
• Model: Transcranial Doppler Sonography 
TCD 
• Location: Charlotte NC, USA 
 
• DiaMon headset can be adjusted in 3 ways to 
accommodate each individual 
• Connection between rear band and side panel 
sits behind the ear, below the occipital bone to 
ensure signal 
• TCD probes should be in contact with head if 
headset has been fitted correctly  
Example Set-up 
 
 
 
• Ensure probe is positioned over the temporal 
window 
• Apply ultrasound gel to probe and/ or 
temporal window 
• Move probe until faint trace appears and then 
make further fine movements to increase trace 
of artery velocity 

Page 25 of 45 
Raw Data 
 
 
 
• Signal quality is indicated by color: the more 
yellow, the stronger the signal 
• Gain and depth adjust can be made in the 
signal window. Gain should sit between 38-44 
for optimal signal output 
• Typical velocities for middle cerebral artery 
(MCA) are ~80 cm/s-1, and ~60 cm/s-1 in 
older populations 
• Typical depth for MCA is 45-55 
 
Data Analysis 
 
 
 
• 
Using a custom macro, the software (Labchart) 
identifies the peak of the ECG R- wave and the 
peak of the second derivative of each pressure 
waveform (corresponds to foot), then calculate the 
transit time (TT) between said peaks. 
• 
Pulse wave velocity (PWV) = distance (L) / TT 
Data Reduction 
 
 
• Measures of brain PWV, MCA velocity, static 
CA and neurovascular coupling will be  
collected from this device are continuously 
recorded during a testing session 
• Data reduction for this protocol will require 
the selection of 30s epochs at specific time 
points to observe how PS and MS are 
impacting measures of CVH 
• In the picture to the left, a researcher selected 
a time period to report the data collected 
during this time. 
• A strict protocol will be followed to ensure 
that selection of 30s epochs are synchronized 
amongst 
all 
measures 
and 
research 
participants to prevent bias. 
 
 
 
 
 
 
 
 
 
 
 

Page 26 of 45 
Device 3: Non-Invasive Blood Pressure (NIBP) 
 Device 
 
 
 
• Company: ADInstruments 
• Model: 
Human 
NIBP 
Nano 
System, 
Finapress 
• Location: Sydney, Australia 
• Single 
cuff 
contains 
an 
infrared 
photoplethysmographic (940 mmHg) sensor 
and inflatable cuff. 
• PPG sensor continuously captures the pulse 
waveform. 
• Inflatable cuff used to calibrate waveform 
• Height 
correction 
unit 
corrects 
for 
hydrostatic pressure changes due to change in 
hand position relative to the heart.  
Example Set-up 
 
 
 
• Device placed between distal and proximal 
inter-phalangeal joint of the index finger. 
• Wrist apparatus is strapped to the individual’s 
non-dominant wrist 
Raw Data 
 
 
• Data captured using a data acquisition system (PowerLab). 
• Also displayed are electrocardiogram (ECG) signals captured by PowerLab.  

Page 27 of 45 
Data Analysis 
• LabChart 8 can perform real time or offline function, including data averaging, peak 
detection, derivates etc.  
Data Reduction 
 
 
 
 
• All measures collected from this device are 
continuously recorded during a testing 
session 
• Data reduction for this protocol will require 
the selection of 30s epochs at specific time 
points to observe how PS and MS are 
impacting measures of CVH 
• In the picture to the left, a researcher selected 
a time period to report the data collected 
during this time. 
• A strict protocol will be followed to ensure 
that selection of 30s epochs are similar 
amongst 
all 
measures 
and 
research 
participants to prevent bias. 
 
Device 4: Doppler Ultrasound 
 Device 
 
 
 
• Company: GE Healthcare 
• Model: Logiq P6 
• Location: Chicago, IL 
• An 11L linear probe will be used for this 
protocol 

Page 28 of 45 
Example Set-up 
 
 
 
• Measurement of blood flow through the 
carotid artery using a linear probe 
• Probe is placed parallel to the blood vessel 
• 10s videos of the common carotid, vertebral, 
and internal carotid arteries will be collected 
at each designated time point in the protocol 
• Participants will be instructed to hold one’s 
breath for the duration of the recording to 
ensure a clear video will be captured of the 
observed artery 
Raw Data 
 
 
 
 
• Ultrasound videos are recorded at 30 Hz 
using an external video capture system 
• Debut Professional (v4.0, NCH Software®) 
is used to record the present image on the 
Ultrasound device for later analysis 
• Image to the left shows an example video 
captured by this software 
Data Analysis 
 
• The captured videos will be analysed offline 
using specialized image analysis software 
(FMD Studio®,) shown to the left.  
• The software measures and exports second-
by-second diameters and blood velocities 
• Cardiovascular 
Suite 
(Quipu, 
v3.5.3) 
analyzes raw ultrasound footage 
• Coordination 
with 
ECG will 
produce 
measures of blood flow 
Data 
Reduction 
 

Page 29 of 45 
 
 
• Lab-designed macro cleans raw data output generated by the video analysis software 
• Macro is capable of calculating blood flow along with other measures that define the 
flow profile of the observed artery 
 
Device 5: Continuous Wave Near-Infrared Spectroscopy with Spatially Resolved 
Spectroscopy (cwNIRS-SRS or NIRS) 
 Device 
 
 
 
• Company: Artinis Medical Systems 
• Model: Portalite 
• Location: Amsterdam, Netherlands 
• Two probes will be used in this protocol 
o Cerebral perfusion: placed over 
the frontal cortex 
o Lower-limb perfusion: placed 
over the muscle belly of the 
gastrocnemius 
Example Set-up 
 
 
 
• Researchers 
will 
follow 
manufacturer 
guidelines on appropriate probe placement 
• An opaque cloth (not shown) will be wrapped 
over the probe to prevent the influence of 
ambient light on infrared signals 
• For NIRS probe placement over the 
gastrocnemius: An 11L linear probe from the 
Logiq 6 ultrasound device will be used to 
ensure the NIRS device is not placed over a 
blood vessel. 
• The ultrasound device is only be used to 
ensure the probe is being placed only over the 
muscle belly of the gastrocnemius 

Page 30 of 45 
Raw Data 
 
 
 
• Oxysoft, a proprietary software system developed by Artinis Medical Systems, will be 
used to collect and display raw data 
• Displayed are three signals each for total hemoglobin (greens), oxygenated hemoglobin 
(reds), and deoxygenated hemoglobin (blues) 
 
 
Data Analysis 
 
 
• The NIRS devices emits three separate 
wavelengths to capture different depths of the  
• 
The spatially resolved signal working with 
the probe is capable of estimating absolute 
values for each variable used in this protocol 
Data Reduction 
 
 
 
• A lab-designed macro will export the raw 
data to an Excel sheet  
• Column 1 represents the time point  
• A researcher will then select 30s sections for 
specific time points during the experimental 
conditions (e.g. immediately post-MAT, 5 
minutes post-MAT) 
 

Page 31 of 45 
 
Device 6: ActiGraph 
 Device 
 
 
 
• Company: ActiGraph 
• Model: wGT3X-BT 
• Location: Pensacola, FL 
 
Example Set-up 
 
 
 
 
 
• Device is worn on the non-dominant wrist 
• To maintain internal validity subjects will be 
asked to complete a sleep journal that details 
when the ActiGraph device is being worn, 
when the participant goes to sleep, and when 
he/she awakens. 
• 
This information will only be used to validate 
the adherence of the participant to pre-test 
behaviors: Waking up at relatively the same 
time of the morning, no vigorous exercise 
24hr prior 

Page 32 of 45 
Raw Data 
 
 
 
 
 
• Upper image: 24 hour observation of counts 
of physical activity and ambient light 
throughout several days 
• Lower image: More detailed view of physical 
activity counts. 
 
 
Data Analysis 
 
 
 
• Trained researchers will use the returned 
sleep journal to set cut points for the 
beginning and end of sleep periods of the 
research participant 
• This process allows more accurate reports of 
ambient physical activity before and during 
experimental sessions 
Data Reduction 
 
 
• Data can be reduced on an hourly or daily basis 
• Accelerometry data separates daily ambulation into different categories: sedentary, 
light, moderate, and vigorous 
 
 
DEVICES 7 & 8: Data Acquisition (PowerLab) and Data Processing (LabChart) 
 Device 
 
 
 
• Company: ADInstruments 
• Model: PowerLab 8/35 
• Location: Sydney, Australia 
• Device will work in conjunction with 
LabChart software and NIBP cuff 

Page 33 of 45 
Example Set-up 
 
 
 
• 
Example set-up shown to an in the image to 
the left. Shown in this example: 
o Upper left: EKG 
o Bottom PowerLab Unit: TCD inputs 
for eventual display on LabChart 
Raw Data 
 
 
 
• 
Shown is LabChart receiving the signals 
interpreted by the PowerLab system  
 
 
Data 
Analysis 
• LabChart 8 can perform real time or offline function, including data averaging, peak 
detection, derivates etc. 
Data Reduction 
 
 
 
 
• After the completion of a data collection set, 
summarized data is calculated by the 
software 
• Data will be exported to lab Excel files for 
comparison amongst subjects and final 
analysis 
 
 

Page 34 of 45 
4  
STATISTICAL CONSIDERATIONS  
4.1  
Study Outcomes 
The study outcomes are listed below. 
 
The primary outcomes are: 
• Brain PWV 
• Brachial-femoral PWV 
 
Note: All power calculations for this protocol are based on measures from PWV 
 
The secondary outcomes are: 
• Executive function 
• Neurovascular coupling 
• Carotid-femoral PWV 
• Augmentation index 
• Femoral-ankle PWV 
Mechanistic and descriptive outcomes are: 
• Cerebral perfusion 
• Total brain blood flow 
• Middle cerebral artery velocity 
• Static cerebral autoregulation 
• Baroreflex sensitivity 
• Heart rate variability 
• Heart rate 
• Peripheral diastolic blood pressure 
• Peripheral systolic blood pressure 
• Tissue saturation index 
• Venous pooling in the gastrocnemius 
• Bouts of physical activity 
• Sleep duration 
 
4.2  
Statistical Analysis 
All statistical procedures will be competed with SPSS Statistics version 25.0 (SPSS, IN., 
Durham, NC, USA) and Jamovi v0.9 (Jamovi, UK). The α-level will be set a prior at 0.05 for all 
statistical procedures. Descriptive statistics and mechanistic outcomes will be collected to 
compare similarities amongst the sample group and to eliminate the potential for extraneous 
covariates. Answering the research question will require a linear multi-level mixed model. 
Statistical models will observe for changes in the primary and secondary outcomes within 
conditions (Baseline, Post-PS, Post-MS) and between conditions (CON vs. SMS at similar time 
points). Mechanistic outcomes will also be used to assist in discussions of physiological 
responses to MS and PS. Effect sizes will be calculated as Cohen’s d, where <0.20 is considered 
to be a small, > 0.20 to < 0.50 a moderate, and > 0.60 a large effect. For the mixed models 
Cohen’s d will be calculated as the effect of condition (β) or time (β) from linear mixed models 
divided by the baseline SD. Raw data will be presented as mean [standard deviation] and mixed 
model data are presented as mean [95% confidence interval]. 

Page 35 of 45 
4.3  
Tabulation of Results 
Mean and variance will be tabulated after raw measures taken in triplicate are entered into a lab-
created template.   
 
Table 4. Methods to describe result summarization for this experimental protocol. 
Abbreviations: X, Mean/SD will be calculated for this result; 1, Measure is reported as a single 
value from each experimental visit; n/a, Measure is not being taken at this timepoint. 
 
4.4  
Missing Values 
Subjects with missing data will be included in the analysis. The linear mixed-effects model is 
well-designed for coping with incomplete longitudinal data. Reasons for non-complete data sets 
will be recorded, and available values will be compared against the population estimates to 
ascertain risk of bias. 
 
 
 
 
 
Baseline 
Post-PS 
Post-MS 
Device 
Outcomes 
X 
SD 
X 
SD 
X 
SD 
VICORDER
® 
bfPWV 
X 
X 
X 
X 
X 
X 
 
cfPWV 
X 
X 
X 
X 
X 
X 
 
faPWV 
X 
X 
X 
X 
X 
X 
 
CBPs 
X 
X 
X 
X 
X 
X 
 
Peripheral BPs 
X 
X 
X 
X 
X 
X 
 
Augmentation 
index 
X 
X 
X 
X 
X 
X 
TCD 
Brain PWV 
X 
X 
X 
X 
X 
X 
 
MCA velocity 
X 
X 
X 
X 
X 
X 
 
Static 
cerebral 
autoregulation 
X 
X 
X 
X 
X 
X 
 
Neurovascular 
coupling 
X 
X 
X 
X 
X 
X 
NIBP 
Baroreflex 
sensitivity 
X 
X 
X 
X 
X 
X 
 
Heart 
rate 
variability 
X 
X 
X 
X 
X 
X 
 
Heart rate 
X 
X 
X 
X 
X 
X 
Ultrasound 
Cerebrovascular 
BF 
1 
X 
1 
X 
1 
X 
NIRS 
Cerebral perfusion 
X 
X 
X 
X 
X 
X 
 
Tissue saturation 
index 
X 
X 
X 
X 
X 
X 
 
Venous pooling 
X 
X 
x 
X 
x 
X 
Cognitive 
Tests 
Executive 
Function 
n/a 
n/a 
n/a 
n/a 
1 
X 

Page 36 of 45 
4.5 Quality Control 
For a given outcome all measurement and analysis will be conducted by a single observer. At the 
start of the study, the first three sets of data collected by the first three research participants will 
be checked by an independent observer.  
 
At the conclusion of the study, a random selection of 10% of the data sets (e.g., all data from 2 
participants) will be re-scored by an independent observer and used to calculate inter-observer 
reliability. 
 
4.6  
Sample Size 
Calculations to determine minimum sample size will be based on central vascular health 
outcome, aortic pulse wave velocity (PWV). We will conduct a power calculation based on 
reported normal values for the primary outcome, central PWV. Healthy people aged 40-49 
maintain an average PWV of 7.5 m/s. For the current study, we will opt to sample based on a 
conservative change score of 1 m/s. We will also use a conservative usual error of 1.25 m/s 
based on reported data. Using magnitude-based inference, to estimate the sample size required to 
detect the smallest detrimental (or beneficial) effect in a cross-over study, with the maximum 
chances of a type 1 and 2 error set at 5% (i.e. very unlikely), approximately eighteen participants 
will be required. 
 
4.7  
Statistical Support 
Statistical expertise, study coordination and statistical computations for research database 
management (RDM) and data analysis will be provided by the research team. Advised by Lee 
Stoner, PhD. If needed, the faculty advisor will submit a request to NCTracs. 
 
 
 

Page 37 of 45 
5  
Participant Screening, Withdrawal & Completion5 
n failure procedures 
Participants will not be able to participate if they do not meet the inclusion criteria. The 
acceptability of participants’ potential recordings from each device will be made during the 
familiarization visit. 
 
5.2  
Participant withdrawal 
If a participant decides to withdraw from the study all efforts should be made to complete and 
report study assessments as thoroughly as possible. The investigator will contact the participant 
by telephone or through a personal visit to establish the reason for the study withdrawal. A 
complete final evaluation at the time of the patient’s study withdrawal should be made with an 
explanation of why the patient is withdrawing from the study. If the reason for removal of a 
patient from the study is an adverse event, the principal specific event will be recorded on study 
data collection forms. If the reason for withdrawal or removal is not an adverse event, the nature 
of the reason will be recorded on the study data collection forms. 
 
5.3  
Participant Completion 
Upon completion of the study, participants will be thanked, and the results discussed. 
 
 

Page 38 of 45 
6  
SAFETY MONITORING & MANAGEMENT 
 
6.1  
Adverse Event Risk 
Investigators leading this project are knowledgeable of clinically healthy values for BP, cardiac 
output, and other experimental outcomes. It is anticipated the collection of values of clinical 
concern will be rare as those with a diagnosis of cardio-metabolic diseases will be excluded from 
this study entirely. However, in the event of clinically meaningful cardiovascular values, the 
participant will be notified of his or her results and consultation with faculty advisor, Dr. Lee 
Stoner will be completed on further action, if needed. It is ultimately the decision of the subject to 
follow through with action after gaining knowledge and receiving advice on his or her unfavorable 
cardiovascular health.  
The devices used in this study are noninvasive. There are no known severely adverse events that 
have occurred with use of the stated devices. 
 
VICORDER® - The system requires the placement of pressure cuffs over several arteries for 
the collection of PWV/A data. Pressure cuffs will only be inflated underneath a level of 65 
mmHg. Physical harm or discomfort is unlikely and include, but are not limited to: 
 
• Risk 1: Discomfort/unease: Infrequent (1 – 10%)  – Application of a slight pressure over 
the carotid artery may impose a sense of unease for the participant. However, the light 
pressure used for this experimental protocol will in no way significantly damage 
cardiovascular structure or place the participant in danger. Investigators will make certain 
that communication on the procedures during testing session are clearly conveyed to the 
participant for comfort and safety.  
 
Near-infrared spectroscopy (NIRS): Risk of injury or discomfort is extremely low due to this 
device. Possible physical harms are, but not limited to: 
• Risk 1: Eye damage/irritation: Rare (<1%) – Please do not, at any point, stare into the 
light emitted from the NIRS probe 
• Risk 2: Skin heating and irritation: Rare (<1%) – Wearing the NIRS probe for extended 
periods of time at once can theoretically lead to a warm feeling at the area where the 
probe is placed. However, this risk is minimal because the light emitted from this probe is 
not powerful enough to heat the skin. If you let the investigators know of any discomfort 
due to the probe, we will follow manufacturer guidelines to ensure the device is 
functioning correctly. 
Transcranial Doppler (TCD): Data collection from this system requires the affixation of a 
headpiece to the participant. Risk of injury due to this device is extremely low. Possible harms 
may include, but are not limited to: 
• Risk 1: Mild headache: Infrequent (1 – 10%)  – High quality data from this device 
requires the placement of the probe over the middle cerebral artery (MCA) and posterior 

Page 39 of 45 
cerebral artery (PCA). The slight pressure applied to the area may be slightly 
discomforting and unusual for the participant. 
6.2 Responding Adverse Events  
In the unlikely event that a subject sustains an injury throughout the duration of the study, they 
will be referred to the appropriate medical personnel on the UNC campus (i.e. Campus Health 
Services or the UNC Hospitals Emergency Room). In the occurrence of a rare adverse event, all 
members of the research team are CPR/AED certified so that they can provide the proper care to 
the Participant. A member of the research team will be with the Participant the whole time while 
in the research lab and 1-2 members of the research team will be present during each exercise 
test. If deemed necessary, emergency medical services will be contacted. 
 
6.3 Confidentiality  
Limiting the number of research team members in the laboratory during any testing session will 
minimize breach of confidentiality. By needing key card access to the laboratory, we are limiting 
the number of individuals not on the research team who have access to the lab. Those who do 
have key card access are exercise physiology professors, Ph.D. candidates, and master’s 
candidates, and selected undergraduate students who are directly associated with the study and 
have performed all necessary trainings regarding sample handling, laboratory procedures, and 
confidentiality. All participants within the study are coded with an individual ID and no names 
will be identified in any document besides a master key document. This master key document 
will be kept in a locked drawer in the Applied Physiology Research Lab. No subjects will be 
identified in any report or publication of this study. 
 
6.4 Participant Withdrawal  
If a subject fails to comply with pre-testing guidelines, their allocated session will be 
rescheduled. If a subject fails to comply with pre-testing guidelines for a second time, they will 
be withdrawn from the study. Any subjects included in the study may withdraw without penalty 
at any point and will be reminded of their right to do so before the commencement of data 
collection. 
 
 
 

Page 40 of 45 
7 
DATA COLLECTION AND MANAGEMENT 
Management of the research data for this study will involve collection, entry, processing, 
storage, retrieval, archival, distribution and documentation of information collected according to 
a written protocol. The overall strategy for quality assurance in data management will be of a 
professional level as described in the following sections. 
 
7.1  
Quality Assurance 
The PI will allocate adequate time to monitor the study to ensure quality and integrity of data 
collected. The PI will review study files, regulatory documents, and consent forms The PI will 
have overall responsibility for ensuring quality in the data and in the procedures that produce the 
data. In addition to the master protocol document, written guidelines and detailed procedure 
manuals may be needed for uniform adherence to the detailed intent of the protocol. The written 
record of how the study was performed will be completed prior to commencement of recruitment 
and will be updated during the course of the study. The PI will have overall responsibility for the 
definition and production of the documents necessary to describe all aspects of the study in 
sufficient detail to ensure the study can be conducted in a scientifically sound, standardized 
manner. The PI will be assisted by members of the research team in monitoring adherence to 
protocol. 
 
7.2  
Database Security 
The database will be created within centralized files and maintained on University approved; 
password protected, encrypted, shared research drives in the orthopedic surgery department and 
viewed only on approved devices by approved study personnel. Collected data will be kept in a 
locked cabinet in the Applied Physiology Lab in Fetzer Hall on the University of North Carolina 
at Chapel Hill campus. Only members of the research team will have keys for this cabinet. All 
shared files between the research team will be stored on a UNC file requiring an Onyen Login 
and either a UNC computer or UNC campus VPN. 
 
7.3  
Confidentiality 
Research data will be identified only by study identification numbers (IDs). These study IDs will 
be used to maintain relationships in the data between various tables. All consent forms and any 
paper data collection instruments will be stored in a locked cabinet in a secure location. The list 
identifying subjects with their contact information will be kept separate from the scientific study 
data. No participants will be identified in any report or publication of this study. Participants will 
be identified via identification code for data collection purposes. All electronically collected data 
will be stored on a computer that only team members Onyen password will allow access to study 
information. This computer will be located in the Graduate Student Office of the Applied 
Physiology Laboratory at the University of North Carolina at Chapel Hill. Data analysis 
procedures will be performed on secure computers within Fetzer Hall, with which password is 
required to access. 
 
7.4  
PHI Security 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). 
Procedures to maintain privacy and confidentiality will be followed rigorously. Each research 

Page 41 of 45 
subject will be assigned a study identification number (ID). The scientific study data will be 
stored in a digital file that does not include patient identifiers or personal health information 
(PHI). A separate data file will contain identifiers and PHI. A master file linking patient 
identifiers to the study data will be kept under lock and key by principal investigator. The 
database will be password protected and will be stored on servers housed in a secure location. 
 
7.5  
Risk of Deductive Disclosure 
We will minimize the risk of unauthorized persons using the database to figure out a subject's 
identity and responses. 
 
7.6  
Data Collection 
Data collection will adhere to precise written instructions and will be monitored to ensure 
adherence to the protocol.  
 
7.7 
Data Entry 
All data entered into the database will be verified by comparing the original source values to the 
values in the database. We will perform double data entry, where one person will enter data into 
a spreadsheet under Tab1. A second person will enter data under Tab 2. A third tab (Tab 3) 
represents the difference in Tab 1 and 2. If values are not 0, the second person will find and fix 
data. 
 
7.8  
Data Editing 
The PI will be responsible for reviewing data-monitoring results and investigating questions 
raised (i.e., queries) about remarkable or questionable data values. 
 
7.9  
Database Documentation 
The names of the variables and their valid ranges or categorical values will be listed in a “data 
book”. Documentation will also include an index of computer programs and an index of reports. 
All programming for statistical computation will include comments providing internal 
documentation. 
 
7.10  Pilot Testing of Operations 
All aspects of data management and project operation will be pilot tested prior to the 
commencement of the study in order to verify adequacy of the methods, materials and systems 
prepared. Every clinical study collects such pilot data –either intentionally prior to 
commencement of the study, or unintentionally after recruitment has begun. 
 
 
 
 

Page 42 of 45 
8 
RECRUITMENT STRATEGYRECRUITMENT STRATEGY 
8.1 
Recruitment Strategies 
Participants will be recruited via email, flyer and/ or workplace presentation from working class 
individuals in the Raleigh-Durham-Chapel Hill area. Given that we are targeting a fairly general 
population, and only a limited number of participants are being recruited, we believe there is a 
high likelihood of having access to the projected number of individuals. 
 
8.2  
Recruitment Personnel 
Recruitment will be conducted by the PI or the trained master’s students listed on this 
application. The team will not recruit students who are currently enrolled in a class they are 
responsible for. 
 
8.3  
Protection of Privacy 
During the recruitment process potential subjects will not be required to confirm their interest in 
the presence of individuals who are not part of the research team. 
 
8.4 
Contacting Participants 
Following the provision of the study information, subjects will be encouraged to contact the 
principal investigator via telephone or email to discuss their involvement or arrange for a 
face-to-face discussion to take place on a separate occasion. 
 
8.4 
Efforts to ensure equal access to participation among women and minorities 
Strong efforts will be made to ensure equal access to participation among all populations. All 
ethnicities have equal access to these places and will thus be given equal access to volunteer as 
subjects. Pregnant women will be excluded from the initial study to limit the variation seen 
within subjects. 
 

Page 43 of 45 
 
 
9 
STUDY MANAGEMENT/ CONSENTSTUDY MANAGEMENT/ CONSENT  
9.1 Consent and Institutional Review Board (IRB) Approval 
Before recruitment and enrollment into this study, the patient will be given a full explanation of 
the study. A member of the research team will cover each section of the consent form in detail 
with the subject. At the end of each section, the research team will ask the subject if he or she has 
any questions. Once the entire consent form is described to the subject, the subject will be 
reminded that they may withdraw from the study at any time without repercussion. After all 
questions have been exhausted and the subject agrees to participate, the subject will sign the 
consent form in the presence of a research team member. Data collection will begin only if the 
subject has signed an informed consent form. 
 
9.2 
Efforts to Minimize Influencing the Participant’s Decision to Participate 
During screening and familiarization, the subject will be allowed to ask any question about the 
study while the consent form is being explained. The subject will sign the consent form after it 
has been described in full and all study-related questions answered. If subjects need additional 
time to decide, it will be granted. It will be made clear to the subject that signing of the consent 
form does not bind the subject to participation in the study and if at any time he or she wishes to 
withdraw from the study, he or she may do so without repercussion. 
 
9.2 Required Documentation 
Before the study can be initiated at any site, the following documentation will be placed on file at 
the University of North Carolina: 
• 
A copy of the official IRB approval letter for the protocol and informed consent 
• 
A copy of the IRB-approved consent form 
 
9.3 Adherence to the Protocol 
Except for an emergency situation in which proper care for the protection, safety, and well-being 
of the study patient requires alternative treatment, the study shall be conducted exactly as 
described in the approved protocol.   
 
9.4 Emergency Modifications 
Members of the research team will inform participants of the study protocol and any potential 
risk. They will then sign an informed adult consent form included in the IRB. As mentioned 
previously, subjects will be properly treated and cared for in the unlikely event of an emergency 
or adverse reaction to any of the noninvasive treatments or venous blood draws.  
 
 

Page 44 of 45 
10 
REFERENCESREFERENCES  
 
1. 
Owen, N., Healy, G. N., Matthews, C. E. & Dunstan, D. W. Too much sitting: The 
population health science of sedentary behavior. Exerc. Sport Sci. Rev. (2010). 
doi:10.1097/JES.0b013e3181e373a2 
2. 
Ng, S. W. & Popkin, B. M. Time use and physical activity: A shift away from movement 
across the globe. Obes. Rev. (2012). doi:10.1111/j.1467-789X.2011.00982.x 
3. 
Dishman, R. K. et al. Neurobiology of exercise. Obesity (2006). doi:10.1038/oby.2006.46 
4. 
Siddarth, P., Burggren, A. C., Eyre, H. A., Small, G. W. & Merrill, D. A. Sedentary behavior 
associated with reduced medial temporal lobe thickness in middle-aged and older adults. 
PLoS One (2018). doi:10.1371/journal.pone.0195549 
5. 
Edwards, M. K. & Loprinzi, P. D. The Association Between Sedentary Behavior and 
Cognitive Function Among Older Adults May Be Attenuated With Adequate Physical 
Activity. J. Phys. Act. Heal. (2016). doi:10.1123/jpah.2016-0313 
6. 
Hamer, M. & Stamatakis, E. Prospective study of sedentary behavior, risk of depression, 
and 
cognitive 
impairment. 
Med. 
Sci. 
Sports 
Exerc. 
(2014). 
doi:10.1249/MSS.0000000000000156 
7. 
Antle, D. M., Cormier, L., Findlay, M., Miller, L. L. & Côté, J. N. Lower limb blood flow 
and mean arterial pressure during standing and seated work: Implications for workplace 
posture recommendations. Prev. Med. Reports 10, 117–122 (2018). 
8. 
Credeur, D. P. et al. Impact of Prolonged Sitting on Peripheral and Central Vascular Health. 
Am. J. Cardiol. (2018). doi:10.1016/j.amjcard.2018.10.014 
9. 
Thosar, S. S. Prolonged Sitting, Oxidative Stress and Vascular Function. (2014). 
10. 
Morishima, T. et al. Prolonged sitting-induced leg endothelial dysfunction is prevented by 
fidgeting. Am. J. Physiol. - Hear. Circ. Physiol. 311, H177–H182 (2016). 
11. 
Restaino, R. M. et al. Endothelial dysfunction following prolonged sitting is mediated by a 
reduction in shear stress. Am. J. Physiol. - Hear. Circ. Physiol. 310, H648–H653 (2016). 
12. 
Morishima, T., Restaino, R. M., Walsh, L. K., Kanaley, J. A. & Padilla, J. Prior exercise 
and standing as strategies to circumvent sitting-induced leg endothelial dysfunction. Clin. 
Sci. (Lond). 131, 1045–1053 (2017). 
13. 
Ogita, H. & Liao, J. K. Endothelial function and oxidative stress. Endothel. J. Endothel. 
Cell Res. 11, 123–132 (2004). 
14. 
Thosar, S. S., Bielko, S. L., Mather, K. J., Johnston, J. D. & Wallace, J. P. Effect of 
prolonged sitting and breaks in sitting time on endothelial function. Med. Sci. Sports Exerc. 
47, 843–849 (2015). 
15. 
Thosar, S. S., Bielko, S. L., Wiggins, C. C. & Wallace, J. P. Differences in brachial and 
femoral artery responses to prolonged sitting. Cardiovasc. Ultrasound 12, 1–7 (2014). 
16. 
Chida, Y. & Steptoe, A. Greater cardiovascular responses to laboratory mental stress are 
associated with poor subsequent cardiovascular risk status: a meta-analysis of prospective 
evidence. Hypertens. (Dallas, Tex.  1979) 55, 1026–32 (2010). 
17. 
Mehra, V. C., Ramgolam, V. S. & Bender, J. R. Cytokines and cardiovascular disease. J. 
Leukoc. Biol. 78, 805–18 (2005). 
18. 
Barnes, A. S. The epidemic of obesity and diabetes. Cardiovasc. Dis. women 38, 142–144 
(2011). 
19. 
WHO. Global action plan for the prevention and control of noncommunicable diseases 
2013-2020. World Heal. Organ. 102 (2013). doi:978 92 4 1506236 

Page 45 of 45 
20. 
Benjamin, E. J. et al. Heart Disease and Stroke Statistics’2017 Update: A Report from the 
American Heart Association. Circulation 135, (2017). 
21. 
Dalgleish, T. et al. Cardiovascular Disease: A Costly Burden for America Projections 
Through 2035. J. Exp. Psychol. Gen. 136, 23–42 (2007). 
22. 
Schulz, K. F., Altaian, D. G. & Moher, D. CONSORT 2010 Statement: Updated guidelines 
for reporting parallel group randomized trials. Japanese Pharmacology and Therapeutics 
(2018). 
23. 
Bowie, C. R. & Harvey, P. D. Administration and interpretation of the Trail Making Test. 
Nat. Protoc. (2006). doi:10.1038/nprot.2006.390 
24. 
Abrahams, S. et al. Verbal fluency and executive dysfunction in amyotrophic lateral 
sclerosis (ALS). Neuropsychologia (2000). doi:10.1016/S0028-3932(99)00146-3 
25. 
Hoiland, R. L. et al. Indomethacin-induced impairment of regional cerebrovascular 
reactivity: 
Implications 
for 
respiratory 
control. 
J. 
Physiol. 
(2015). 
doi:10.1113/jphysiol.2014.284521 
26. 
Stone, K. J., Fryer, S. M., Ryan, T. & Stoner, L. The validity and reliability of continuous-
wave near-infrared spectroscopy for the assessment of leg blood volume during an 
orthostatic challenge. Atherosclerosis 251, 234–239 (2016). 
27. 
Hopkins, W. G., Marshall, S. W., Batterham, A. M. & Hanin, J. Progressive statistics for 
studies in sports medicine and exercise science. Med. Sci. Sports Exerc. 41, 3–12 (2009).