Sex Differences in Chronic and Acute Vascular Responses to Aerobic Exercise in Older Adults
Phase: NA
Conditions: Aging
Sponsor: University of Florida

Official title:  
Sex Differences in Chronic and Acute Vascular Responses to Aerobic Exercise in Older Adults 
 
NCT #:  
NCT04128215 
 
Document date:  
08/30/2022 
 
 
 

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3. Abstract 
Cardiovascular disease (CVD) is the leading cause of death in the U.S. in both men and 
women. Although biological sex is an important determinant of CVD pathophysiology, a 
major knowledge gap remains regarding sex differences in CVD prevention and 
treatment. A key early event in CVD development is endothelial dysfunction, 
characterized by impaired flow-mediated dilation (FMD). Aging progressively impairs 
endothelial function which independently predicts CVD progression and events. 
Therefore, strategies to optimize endothelial function in aging are clinically important. 
Regular aerobic exercise ameliorates endothelial dysfunction in healthy older men, but 
the data in healthy postmenopausal women are inconsistent with many studies showing 
no effect. The mechanisms underlying these sex disparities are unknown. Higher 
intensity exercise is thought to be required for chronic endothelial adaptations in 
postmenopausal women, but this has not been investigated. We have developed a 
novel exercise strategy by adapting high-intensity interval training (HIIT) on a non-
weight-bearing all-extremity (NWA) ergometer. We have demonstrated that our 
innovative NWA-HIIT is feasible and safe in older adults. The primary objective of this 
study is to examine sex differences in acute and chronic endothelial responses to NWA-
HIIT in older men vs. postmenopausal women. Our overarching hypothesis is that acute 
and chronic endothelial responsiveness to aerobic exercise is influenced by sex in 
healthy aging and acute endothelial responses to exercise will be predictive of chronic 
endothelial adaptations.  
4. Background 
Cardiovascular disease (CVD) is the leading cause of death in the U.S. in both men and 
women. Although biological sex is increasingly recognized as an important determinant of 
CVD development, progression and outcomes, a major gap remains regarding sex 
differences in CVD pathophysiology, prevention and treatment. A key early event in CVD 
development is endothelial dysfunction, characterized by impairments in flow-mediated 
dilation (FMD)1, 2. In women, the onset of endothelial dysfunction begins a decade later 
than in men, but the rate of decline is steeper3. Aging progressively impairs endothelial 
function which independently predicts CVD progression and events4. Therefore, 
strategies to optimize endothelial function in aging are clinically important but should 
account for the potential impact of sex. 
Aerobic exercise is often prescribed to reduce CVD risks; however, controversy exists 
regarding the optimal exercise prescription for preserving or restoring endothelial function 
in older adults. Moderate-intensity aerobic exercise ameliorates endothelial dysfunction in 
older men, but the data in postmenopausal women are inconsistent, with most studies 
showing no effect (reviewed in Seals et al5). The mechanisms underlying these sex 
disparities in healthy aging are unknown. A recently introduced theory of exercise-induced 
hormesis proposes that an acute exercise bout that generates an optimal level of stress 
can stimulate adaptations to increase resistance against subsequent stressors; if the 
exercise-induced stress does not reach this threshold, then adaptation will not occur. 
Consistent with this theory, higher exercise intensity is thought to be required to induce 
chronic endothelial adaptations in postmenopausal women, but this has not been  

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investigated. High intensity interval training (HIIT) is receiving considerable attention as 
an innovative strategy for cardiac rehabilitation, but data in sedentary aging are limited 6, 
7. HIIT involves short periods of vigorous exercise, mainly “uphill” treadmill walking, 
interspersed by periods of moderate-intensity exercise. However, weight-bearing HIIT is 
often problematic in older adults due to musculoskeletal issues. We have recently 
developed a novel alternative by adapting HIIT to a non-weight-bearing all-extremity 
(NWA) ergometer; our innovative NWA-HIIT allows compensation for lower extremity 
weakness/fatigue. We have established in sedentary older adults that NWA-HIIT is 
feasible, well-tolerated and safe8. Our preliminary data suggest that 8-week NWA-HIIT 
improves endothelial function in older men, but not in postmenopausal women. Our 
preliminary studies also indicate substantial sex differences in acute endothelial 
responsiveness to HIIT; there is a transient attenuation in endothelial function in older 
men following an acute bout of HIIT, but this is absent in postmenopausal women. We 
postulate that in men, NWA-HIIT presents an optimal challenge to endothelial 
homeostasis thereby initiating chronic adaptive responses; in contrast, the stimulus is 
inadequate for chronic adaptations in postmenopausal women. Accordingly, acute 
responses to exercise may provide keys to understanding sex-related differences in long-
term endothelial adaptive responses to exercise. 
Our overarching hypothesis is that acute and chronic endothelial responsiveness 
to aerobic exercise is influenced by sex in healthy aging and acute endothelial 
responses will be predictive of chronic endothelial adaptations. Our experimental 
model of acute and chronic responses to NWA-HIIT presents a unique tool to test this 
hypothesis and explore the underlying mechanisms in healthy sedentary older adults. Our 
translational approach will examine, for the first time, sex differences in endothelial 
responses to exercise at multiple levels spanning from the molecular and cellular to the 
organism level. Identifying the defective adaptive pathways in women may lead to novel 
therapeutic targets. 
 
5. Specific Aims 
1. To study sex differences in chronic endothelial adaptations to aerobic exercise  
2. To study sex differences in acute endothelial responses to aerobic exercise and if acute 
endothelial responses are predictive of chronic endothelial adaptations. 
 
6. Research Plan 
Study design and timing of measures. An equal number of older men and 
postmenopausal women will be enrolled in this study and the same subjects will complete 
Aims 1 and 2. For Aim 1, subjects will complete 8-week NWA-HIIT preceded by an 8-
week control period of normal lifestyle. Therefore, each subject will serve as his/her own 
control and the control period of normal lifestyle will also allow determination of potential 
changes due to measurement error. FMD and other measures associated with Aim 1 will 
be assessed at baseline, and after the 8-week control and 8-week NWA-HIIT (Diagram 
1). FMD and other measures associated with Aim 2 will be assessed at pre-exercise, at 
the end of a single session of NWA-HIIT, and 1-hr and 24-hrs post-exercise (Diagram 2) 
in the untrained and trained state (Diagram 1). Measures in the trained state for Aim 2 
will be completed 72-96 hours after the last chronic exercise session. 

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Study procedures will be performed at the Integrative Cardiovascular Physiology 
Laboratory, Center for Exercise Science, University of Florida. 
 
Exercise intervention. Our HIIT regimen is 
based on a treadmill protocol that has been 
shown to be safe and highly effective in 
improving 
CVD 
risks 
in 
patients 
with 
cardiometabolic disease7, 9. NWA-HIIT will be 
performed on an air-braked ergometer (AD7, 
Schwinn) 4 days/week, for 8 weeks, as we 
have previously published8. To mitigate the 
risk for COVID-19, exercise sessions will be 
performed at the participant’s home and will 
be remotely supervised live via Zoom by a 
member of the Integrative Cardiovascular 
Physiology Laboratory. The participant will be 
provided the following to be used during the 8-week exercise training: 1) an exercise 
ergometer; 2) a Zephyr wearable device (Medtronic); 3) a tablet with the OmniSense 5.0 
software (Medtronic) and Zoom and WiFi capability. Participants who do not have WiFi 
available at their home will be provided with a data plan. A familiarization session will be 
provided for using Zoom and the Zephyr wearable device and Omnisense software. The 
 
 
Diagram 1. Study timeline for Aims 1 and 2 
 
 
Diagram 2. Study timeline for Aim 2 
 
Diagram 3. NWA-HIIT exercise protocol 

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Zephyr Cloud technology allows real-time monitoring of physiological responses during 
exercise including heart rate, breathing rate, body temperature, caloric expenditure, and 
accelerometry. A member of the Integrative Cardiovascular Physiology Laboratory will 
lead each virtual exercise session and will guide the participants through the setup, warm 
up, work out and cool down. 
NWA-HIIT will consist of 4x4-min bouts at 90% of maximal heart rate (HRmax) interspersed 
by 3x3-min bouts at 70% of HRmax (Diagram 3)8. A 10-min warm-up and 5-minute cool-
down at 70% of HRmax will be included. HRmax will be determined during the baseline 
maximal exercise test. Chronic NWA-HIIT will be preceded by a period of familiarization 
as we have previously described8. Based on our extensive experience, on average 5±1 
session is required in sedentary older adults to be able to complete an entire HIIT session. 
The total number of completed exercise sessions by each study participant will depend 
on their ability to perform all-extremity exercise, their initial fitness level and scheduling 
availability for the post-intervention procedures. The number of sessions will be recorded 
and incorporated in data analysis and interpretation.  
 
Study Procedures for Aims 1 and 2  
• Medical history. Subjects will complete a medical history form. 
• Physical exam. A clinician on our team will perform a physical examination. This will 
consist of review of medical history and check of general physical condition including 
examination of the head and neck, heart, lungs, abdomen, nerves, muscles, joints and 
skin. The Allen test which evaluates blood circulation to the hands will be performed 
to screen out subjects for whom intra-arterial cannulation is contraindicated. 
• 24-hour questionnaire. We will ask questions regarding sleep, food, drink, tobacco, 
alcohol, caffeine and medication intake and physical activity over the last 24 hours. 
We will also ask if subjects are experiencing any health problems (e.g., sore throat, 
head cold, upset stomach, etc.). 
• Height. We will measure height using a ruler attached to a wall.  
• Weight. We will measure body weight using an electronic scale.  
• Heart rhythm. We will record heart rhythm.  
• Blood pressure. We will measure blood pressure with a cuff around the ankles. We 
will also measure blood pressure with a cuff around the upper arms.  
• Body composition and circumferences. We will measure waist and hip circumferences 
using a tape measure. We will also measure body composition including body fat and 
fat free weight, as well as bone density, using a non-invasive dual-energy x-ray 
absorptiometry (DXA) scan as we have previously published8. This test requires that 
subjects lie very still on a padded table that gives off low-level X-ray radiation. The 
“arm” of the machine will slowly pass over the body to make the measures. This should 
take less than 10 min. Female participants who are younger than 62 years of age and 
who have not had a hysterectomy, will complete a urine pregnancy test prior to the 
scan. The DXA scan will be prescribed by Dr. Carl Pepine from the Division of 
Cardiology. 

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o To investigate the chronic effects of exercise on body composition and 
circumferences, these tests will be performed: 1) at baseline; 2) after 8 weeks 
of normal lifestyle; and 3) after 8 weeks of exercise training. 
• Physical activity habits. We will ask questions regarding how hard, how often and for 
how long subjects perform a variety of physical activities. We will also ask subjects to 
wear an activity monitor over their hip for 4 days to track their activity. 
o These measures will be performed: 1) at baseline; 2) after 8 weeks of normal 
lifestyle; and 3) after 8 weeks of exercise training. 
• Dietary habits. We will ask questions regarding what subjects ate and drank on the 
previous day.  
o This information will be collected: 1) at baseline; 2) after 8 weeks of normal 
lifestyle; and 3) after 8 weeks of exercise training. 
• Quality of life questionnaire. We will ask questions about perceptions of health and 
ability to perform daily activities.  
o This information will be collected: 1) at baseline; 2) after 8 weeks of normal 
lifestyle; and 3) after 8 weeks of exercise training.  
• Maximal exercise test on treadmill. An incremental maximal exercise test will be used 
to measure maximal oxygen consumption (VO2max) on the treadmill and screen out 
subjects with cardiovascular problems as we have previously published 8. ECG and 
blood pressure responses will be monitored by our experienced study clinicians  and 
oversight will be provided by our cardiologist (Dr. Carl Pepine). VO2max will be 
measured using open-circuit spirometry8. To measure oxygen consumption, subjects 
will breathe through a mouthpiece while wearing a nose clip. The treadmill will begin 
slowly and the speed (pace) will gradually increase based on their walking ability and 
their heart rate. Upon completion of a 6-min warmup, the speed will remain the same 
while the treadmill will slightly tilt every 2 min to produce the effect of going up a small 
hill until they reach their maximal exercise capacity (usually 12 to 15 min excluding 
the warm-up period). At certain times during the test, we will ask them to point to a 
scale which tells us how hard they feel they are working. If they have a problem 
walking on the treadmill they may perform the test on a stationary bicycle. If any 
abnormalities occur during the test, they will be referred to their primary care physician 
for further testing.  
• Maximal exercise test on all-extremity cycle. This test will measure the maximal ability 
to perform all-extremity cycling. Heart rhythm, blood pressure and oxygen 
consumption will be measured throughout the test as described above. Subjects will 
complete a 6-min warmup by performing all-extremity cycling at a moderate pace. 
Upon completion of the warmup, the pace will gradually increase every 2 min until 
they reach their maximal exercise capacity (usually about 12 to 15 min excluding the 
warmup). Prior to this visit they will be given an opportunity to practice all-extremity 
cycling and breathing through a mouthpiece. 

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o To investigate the chronic effects of exercise on maximal exercise capacity, 
this test will be performed: 1) at baseline; 2) after 8 weeks of normal lifestyle; 
and 3) after 8 weeks of exercise training. 
• 6-min walk test. The purpose of this test is to assess the ability to walk for 6 min. The 
test will be completed at the subject’s pace, walking back and forth between 2 cones. 
The objective is to walk as far as possible in 6 min. They will be permitted to slow 
down, to stop, and to rest, as necessary. 
o To investigate the chronic effects of exercise on walking ability, this test will be 
performed: 1) at baseline; 2) after 8 weeks of normal lifestyle; and 3) after 8 
weeks of exercise training. 
• Blood vessel tests. Our primary outcome is brachial artery FMD, an established non-
invasive measure of endothelial function10. FMD will be determined via 
ultrasonography as we have previously described11, 12 and strictly conforming with 
established guidelines13. Arterial stiffness is known to influence FMD, therefore, it will 
be non-invasively measured using the SphygmoCor device as we have previously 
described 14. Measures will be performed while subjects rest supine in a quiet, semi-
darkened, temperature-controlled room. Individual subject data will be coded to 
ensure blinding during data analysis. 
o To investigate the chronic effects of exercise on blood vessel function, these 
tests will be performed: 1) at baseline; 2) after 8 weeks of normal lifestyle; and 
3) after 8 weeks of exercise training. 
o To investigate the acute effects of HIIT on blood vessel function, these tests 
will be performed: 1) at rest; 2) at the end of a HIIT session; 3) after 1-hour 
recovery; and 4) after 24-hours recovery. To examine how chronic exercise 
training affects the acute responses of exercise, these tests will be performed 
at the beginning and end of the 8 weeks of exercise training. 
o To investigate the acute effects of maximal exercise on blood vessel function, 
these tests will be performed: 1) at rest; 2) at the end of the maximal exercise 
test on treadmill (described above); 3) at the end of the maximal exercise test 
on all-extremity cycle (described above); and after 1-hour recovery. To 
examine how chronic all-extremity exercise training affects the acute 
responses to maximal exercise test on all extremity cycle, these tests will be 
performed at the beginning and end of the 8 weeks of exercise training. 
• Nitroglycerin test. Endothelium-independent dilation, a measure of vascular smooth 
muscle responsiveness to a nitric oxide donor (0.4 mg nitroglycerin sublingual tablet; 
an FDA approved drug which causes blood vessels to relax) will be determined as we 
have previously described 15. Brachial artery images will be recorded using 
ultrasonography before nitroglycerin administration and for up to 10 min while subjects 
lie still on their back. Prior to administering nitroglycerin, an intravenous catheter will 
be in place and will remain for up to 30 min after taking the nitroglycerin pill.  
o To investigate the chronic effects of exercise on blood vessel function, the 
nitroglycerin test will be performed: 1) at baseline; 2) after 8 weeks of normal 
lifestyle; and 3) after 8 weeks of exercise training. 

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• Blood draw. The amount of blood drawn over study participation (6-7 months) will total 
25 TBSP, which is less than a whole blood donation which is typically a pint.  Subjects 
will be informed not to donate blood during study participation or within 2 months of 
completion. 
o To perform general blood tests including comprehensive metabolic panel, lipid 
panel, insulin and blood cell count with differential, a TBSP of blood will be 
collected: 1) at baseline; 2) after 8 weeks of normal lifestyle; and 3) after 8 
weeks of exercise training.  
o To investigate the chronic effects of HIIT, 2 TBSP of blood will be collected to 
measure circulating markers of oxidative stress and inflammation and 
endothelial function, vasoconstrictor and sex hormones and to isolate peripheral 
blood mononuclear cells to investigate protein and gene expression of markers 
of oxidative stress and inflammation and endothelial function: 1) at baseline; 2) 
after 8 weeks of normal lifestyle; and 3) after 8 weeks of exercise training. 
o To investigate the acute effects of HIIT, 2 TBSP of blood will be collected to 
measure circulating markers of oxidative stress and inflammation and 
endothelial function and to isolate peripheral blood mononuclear cells to 
investigate protein and gene expression of markers of oxidative stress and 
inflammation and endothelial function: 1) at rest; 2) at the end of a HIIT 
session; 3) after 1-hour recovery from HIIT; and 4) after 24-hours recovery 
from HIIT. To examine how chronic exercise training affects the acute 
responses of exercise, these tests will be performed at the beginning and end 
of 8 weeks of exercise training. 
• Endothelial cell collection and analysis. We have the experience, resources, and 
access to experts to support the proposed endothelial cell measures. The proposed 
study will lay the foundation upon which we will base future studies focusing on 
dissecting in vivo the sex-specific effect of exercise on mechanisms of endothelial 
dysfunction. The procedures for studying endothelial cells have been described in 
detail by Colombo et al16-18, and by our group 11, 19-22. A clinician on our team from the 
Pulmonary and Critical Care Division, Cesar Trillo-Alvarez, MD, who has extensive 
experience with intra-arterial catheterizations (~1500 procedures over 15 years) will 
perform the procedure. He will place a standard 20 gauge arterial catheter with no 
sheath at the radial artery guided by ultrasonography after numbing the area with local 
lidocaine injection. No vasodilators will be used. Once the catheter is secure, four 
small (0.018-inch diameter) sterile J-shaped guidewires will be sequentially advanced 
(~4 inches) through the radial arterial catheter and retracted to collect a small number 
of endothelial cells. Following the cell collection and blood draw, the catheter will be 
removed and hemostasis will be achieved by manual compression of the site. The 
physician will monitor bleeding at 5, 10 and 15 minutes after removal of the catheter 
to assure hemostasis and no hematoma formation. Subjects will not be on 
anticoagulants, therefore normal coagulation time is anticipated. Cells will be 
recovered by rinsing the wires and centrifugation. Endothelial cell protein levels of key 
factors associated with oxidative stress, inflammation, and endothelial function and 
sex hormone receptors will be examined using immunostaining. Detailed procedures 
for immunofluorescence staining and analysis have been described previously by our 

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group 11, 19-22. Endothelial cells will also be analyzed using RNA sequencing to identify 
new pathways that lead to sex differences in endothelial responses to exercise in older 
adults. 
o To investigate the chronic effects of HIIT, cells will be collected and analyzed: 
1) at baseline; 2) after 8 weeks of normal lifestyle; and 3) after 8 weeks of 
exercise training. 
• Protein and mRNA levels in peripheral blood mononuclear cells (PBMCs). Protein and 
mRNA levels will be determined for key factors associated with oxidative stress, 
inflammation, and endothelial function and sex hormone receptors. Ficoll-Paque will 
be used to isolate the mononuclear cells from whole blood by centrifugation. Protein 
levels will be analyzed using Western blots. RNA isolation will be performed using the 
RNAqueous (Ambion) kit and gene expression will be measured using RT-PCR23.  
o To investigate the acute effects of HIIT on protein and mRNA levels, these 
tests will be performed: 1) at rest; 2) at the end of a HIIT session; 3) after 1-
hour recovery; and 4) after 24-hours recovery. To examine how chronic 
exercise training affects the acute responses of exercise, these tests will be 
performed at the beginning and end of the 8 weeks of exercise training. 
 
List of Procedures and Duration by Visit  
Consent (1.5 hrs)  
Informed consent  
Medical history and activity questionnaire 
Visit 1 (1.5 hrs)  
24hr questionnaire and activity monitor pick up 
Height/weight 
Blood pressure 
Visit 2 (1.5 hrs) 
24hr questionnaire 
Weight 
Physical exam 
Blood pressure and heart rhythm 
Maximal exercise test on treadmill 
Pre-exercise and end of exercise: 
   Blood vessel tests 
Visit 3 (1 hr) 
Questionnaires  
Pregnancy test  
Weight, waist/hip circumferences, body composition scan 
6-min walk test 
All-extremity cycling practice 
Visit 4 (1 hr) 
24-hr questionnaire 
Weight 
Blood pressure and heart rhythm 
Maximal exercise test on all-extremity cycle 

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Visit 5 (2.5 hrs)  
24-hr questionnaire 
Weight 
Blood pressure and heart rhythm 
Blood vessel tests 
Nitroglycerin test and intravenous catheter 
Intra-arterial catheter, cell collection and blood draw 
8-week control period: normal lifestyle 
Visit 6 (1 hr)  
Questionnaires and activity monitor pick up 
Pregnancy test  
Weight, waist/hip circumferences, body composition scan 
6-min walk test 
All-extremity cycling practice  
Visit 7 (2.5 hrs)  
24-hr questionnaire 
Weight 
Blood pressure and heart rhythm 
Blood vessel tests 
Nitroglycerin test and intravenous catheter 
Intra-arterial catheter, cell collection and blood draw 
Visit 8 (3 hrs) 
24-hr questionnaire 
Weight 
Maximal exercise test on all-extremity cycle 
Pre-exercise, end and 1-hour post-exercise: 
   Blood pressure and heart rhythm 
   Blood vessel tests 
All-extremity HIIT Practice       
Prior to initiating the 8-week training program, a period of familiarization with all-extremity HIIT 
exercise will be provided. During this time, the exercise duration and pace will gradually increase 
with each session until subjects are able to complete the HIIT protocol. 
Visit 9a (3 hrs) 
24-hr questionnaire 
Weight 
Exercise session on all-extremity cycle 
Pre-exercise, end and 1-hour post-exercise: 
   Blood pressure and heart rhythm 
   Blood vessel tests, intravenous catheter and blood draw  
Visit 9b (1 hr) 
24-hr questionnaire 
Weight 
Blood pressure and heart rhythm 
Blood vessel tests and blood draw 
8-week training period:40 min/session scheduled every week on M, T, Th, F  
 
 

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Visit 10 (2.5 hrs)  
24-hr questionnaire and activity monitor pick up 
Weight 
Blood pressure and heart rhythm 
Nitroglycerin test and intravenous catheter 
Intra-arterial catheter, cell collection and blood draw 
Visit 11 (1 hr)  
Questionnaires 
Pregnancy test 
Weight, waist/hip circumferences and body composition scan 
6-min walk test 
Visit 12a (3 hrs) 
24-hr questionnaire 
Weight 
Exercise session on all-extremity cycle 
Pre-exercise, end and 1-hour post-exercise: 
   Blood pressure and heart rhythm 
   Blood vessel tests, intravenous catheter and blood draw 
Visit 12b (1 hr) 
24-hr questionnaire 
Weight 
Blood pressure and heart rhythm 
Blood vessel tests and blood draw 
Visit 13 (3 hrs) 
24-hr questionnaire 
Weight 
Maximal exercise test on all-extremity cycle 
Pre-exercise, end and 1-hour post-exercise: 
   Blood pressure and heart rhythm 
   Blood vessel tests 
 
These visits will be scheduled over 6-7 months which should alleviate some of the 
burden: 
1) visits 1-5 will be scheduled over 2-3 weeks and will total 7.5 hours; 
2) during the 8-week control period of normal lifestyle there will be no scheduled 
visits;  
3) visits 6, 7, 8, 9a and 9b will be scheduled over 2-3 weeks and will total 10.5 
hours;  
4) during the 8-week exercise training period there will be 4 scheduled exercise 
sessions/week;  
5) visits 10, 11, 12a, 12b and 13 will be scheduled over 2-3 weeks and will total 
10.5 hours.  
 
Sample Size, Statistical Analyses, and Anticipated Results 
Sample size justification. For power analyses, we set α=0.05 and β=0.80 and used the 
smallest effect size (ES) based on our preliminary data on FMD, which is our primary 
outcome. To detect significant sex differences in acute/chronic FMD responses, we will 
need 11 men and 11 women (ES=1.57 for acute and 1.28 for chronic response). To detect 

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significant acute and chronic changes in FMD in response to NWA-HIIT in older men we 
will need 10 men (ES=1.02 for acute and 3.87 for chronic response). We anticipate we 
will need to consent and screen 165 research participants to meet the study inclusion 
criteria and obtain complete datasets in 11 men and 11 women. 
 
Statistical analyses. Our primary outcome will be FMD. For both Aim 1 and 2, we will 
use mixed factorial ANOVA. Post hoc and multiple tests will be corrected using the 
Bonferroni adjustment. Secondary analyses will be performed using ANCOVA to account 
for influence of potential confounders. Exploratory analysis will evaluate the mechanisms 
by which exercise influences FMD and stimulate future mechanistic studies. Using linear 
regression, we will examine whether responses to acute NWA-HIIT are correlated with 
responses to chronic 8-week NWA-HIIT. Analysis of the RNA sequencing data will be 
conducted by the ICBR bioinformatics staff. 
 
HUMAN SUBJECTS  
Human Subject Characteristics  
Subjects will be sedentary men and postmenopausal women recruited from the 
community using advertisements.  Subjects will be screened by our experienced study 
clinicians with oversight by our cardiologist (Dr. Pepine) according to the study 
inclusion/exclusion criteria based on the medical history, physical examination, general 
blood tests and diagnostic maximal exercise test. 
Inclusion criteria 
• adults able to give consent 
• men and women  
• women must be postmenopausal (either natural or surgical)  
• 60 to 79 years of age  
 
Exclusion criteria 
• any relevant cardiovascular diseases (e.g., history of coronary artery bypass 
surgery or angioplasty, or heart failure, myocardial infarction, angina pectoris, 
peripheral arterial disease) 
• myocardial ischemia during maximal graded exercise test 
• major chronic clinical disease (e.g., diabetes, renal or hepatic disease or infection 
with hepatitis B, C, or HIV) 
• seizures, or other relevant on-going or recurrent illness 
• recent (within 3 months) or recurrent hospitalizations 
• systolic blood pressure <100mmHg 
• body mass index > 35 kg/m2 
• >5% weight change in past 3 months or unwilling to remain weight stable during 
study participation  
• use of tobacco products including smoking traditional or e-cigarettes 
• use of hormone replacement therapy in women or men (e.g., estrogen, 
progesterone or testosterone) 

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• regular aerobic exercise training (≥30 min/session and ≥ 3 days/week) 
 
Informed Consent  
Research volunteers who contact the PI’s laboratory in response to our recruitment 
efforts (advertisements) will be asked to complete a phone screening or an online 
RedCap screening questionnaire to determine if they qualify to participate in the study. 
Volunteers who meet the preliminary inclusion criteria and are willing to participate will 
be given the consent form to read prior to obtaining written consent. Informed consent 
will be obtained by the PI, Dr. Christou, or her research assistant, Stephanie Lapierre, in 
a quiet and private area or via a secure password-protected UF Zoom meeting. If 
consent is obtained via Zoom, then the subject’s signature will be obtained using 
REDCap eConsent. All procedures and risks related with participation in the study will 
be described and the subject’s understanding of the study will be confirmed. Any 
questions will be answered prior to obtaining the subject’s written consent. The consent 
form will then be signed by both the subject and investigator as documentation of 
consent. Consent will be documented in the subject’s research file. A copy of the signed 
consent form will be provided to the subject.  
 
Compensation 
Subjects will be compensated a total of $360 for completing visits 5-13 in the form of a 
gift card upon completion of the study. In addition, for every week that a subject 
completes all 4 supervised exercise sessions s/he will be compensated an additional $5 
(e.g., a subject who completes 4 sessions/week for 8 weeks will receive an additional 
$40, thus increasing the total compensation from $360 to $400). Payment will be given 
to subjects within a few weeks following completion of their participation or withdrawal 
from the study. 
 
If subjects do not complete all visits, then compensation will be prorated according to 
the following plan: 
 
Visits 1-4 
Will receive the health screening results at no cost. 
No financial compensation will be provided. 
Visit 5 
$45 
Visit 6 
$10 
Visit 7 
$45 
Visit 8 
$25 
Visit 9a 
$25 
Visit 9b 
$10 
Visit 10 
$75 

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Visit 11 
$10 
Visit 12a 
$45 
Visit 12b 
$20 
Visit 13 
$50 
Exercise sessions 
Will receive supervised exercise training at no cost. 
An additional $5/week will be provided for each week that all 4 
supervised sessions are completed. 
 
Protection of Privacy and Confidentiality 
Subject privacy will be protected by performing research procedures in a private room. 
When use of UF Zoom is needed, a password-protected session will be used and the 
option to record will be disabled. The collection of sensitive information will be limited to 
the amount necessary to achieve the aims of the research, so that no unneeded 
sensitive information is being collected. All subject identities and records will remain 
strictly confidential and will be accessible only by the research team. Whenever feasible 
identifiers will be removed from the study-related information. The names of the 
subjects will not be identified in any publication or presentation arising from these 
studies. Ultrasound images will be labeled by subject ID and will be stored digitally and 
backed-up on tapes. Blood, endothelial cell and peripheral blood mononuclear cell 
samples will also be labeled using subject ID numbers. The samples and tapes will be 
stored in the PI’s laboratory where access is limited to the study personnel. Paper 
copies of data will be stored in a locked cabinet in a secure location accessible only to 
members of the investigative team. Electronic data will be kept on the laboratory server, 
which is accessed by individual user login and passwords. Electronic data will be 
backed-up on the College server which is also password-protected. Real-time exercise 
response data collected live by the Zephyr Omnisense wearable device will be stored 
using subject ID to the Zephyr Omnisense cloud and will be downloaded to the College 
server; to access the cloud and data on the server will require user login and password.   
 
7.  Possible Discomforts and Risks 
• Flow mediated dilation- Inflating the blood pressure cuff on the forearm during 
this procedure may cause a mild to moderate intensity “pins and needles or 
numbing” sensation that goes away as soon as the cuff is deflated.  
• Body composition (DXA) scan- The radiation exposure from each DXA scan is 
equal to about 3 millirem, which is comparable to about 4 extra days of natural 
background radiation to which people in the United States are exposed to 
during their lives.  The risk from this radiation exposure is considered to be 
extremely low when compared to other everyday risks.  
• Maximal graded exercise test- Arrhythmia (<1%); minimal risk of myocardial 
infarction (<0.05%) and death (<0.02%); small risk of syncope and fatigue. 

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• Exercise training- When one begins a new exercise program there is a risk that 
it may lead to fatigue or muscle or joint soreness, or injury. There is a small risk 
of irregular heart rhythm and very rarely heart attack or stroke which could 
potentially lead to death. In general, exercise does not lead to heart problems or 
stroke in individuals who have completed health screening and are free of heart 
disease. To allow monitoring of the exercise responses using the wearable 
device, the location option on the tablet must be turned on. Neither the study 
team nor the company that manufactures the wearable device will have access 
to the location information. But there is a small risk that an unauthorized person 
may get access to this information, while the tablet is in use. 
• Intravenous catheter and blood draw- Discomfort at the site of puncture; 
possible bruising and swelling around the puncture site; rarely an infection or 
blood clot; and, uncommonly, faintness from the procedure.  
• Intra-arterial catheter and cell and blood collection- Discomfort associated with 
lidocaine injection. Discomfort from needle stick and placement of catheter at 
the time it is inserted or after it has been removed. Possible bleeding, bruising, 
and swelling around the puncture site. Rarely local blood clot, infection, 
significant blood loss, nerve or artery damage, tissue injury and damage of 
surrounding tissue and extremity. Pain from compression of puncture site if 
severe bleeding occurs. The risks related to the cell collection are similar to 
those associated with an intra-arterial catheter, although the risk of blood clot, 
infection or blood vessel damage may be slightly greater compared to having 
intra-arterial catheter placement without cell collection.  
• Lidocaine- Lidocaine will be used for numbing the area prior to arterial 
cannulation. Expected sensation of coolness or numbness at injection site; 
likely mild temporary skin irritation including burning, redness or swelling at 
injection site; unlikely but possible reactions include nausea, vomiting, 
dizziness, ringing in ears, nervousness and blurred or double vision; extremely 
rare allergic reaction. 
• Nitroglycerin test- The oral administration of nitroglycerin (0.4 mg sublingual 
tablet) is expected to cause a small transient decrease in arterial blood 
pressure. The subject may experience increased heart rate, lightheadedness, 
tingling on the tongue and in the arms and legs, light headache, and at the 
extreme, fainting. In rare cases heart rate may decrease in response to the 
nitroglycerin pill which can also cause fainting. The dose of nitroglycerin used is 
considered safe and is commonly used in patient populations to relieve angina. 
With any drug used in testing, there is a small risk of an unforeseen life-
threatening allergic reaction. The on-site study clinician will confirm that there 
are no contraindications for nitroglycerin administration (e.g., systolic blood 
pressure <100 mmHg, phosphodiesterase inhibitors including Sildennafil) 
before administering the drug and will monitor the subject until ready to be 
released.  
 
 
 

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Protection Against Risks 
In general, risks will be minimized by: 
• allowing only individuals who meet the inclusion criteria to participate 
• screening for contraindications for nitroglycerin administration, maximal graded 
exercise testing or intra-arterial catheter placement 
• providing clinical supervision, including ECG and blood pressure monitoring, for 
the maximal graded exercise tests and nitroglycerin administration 
• ensuring constant personal monitoring of each experimental session by the 
investigators  
• using only safe, well-established procedures, and having only qualified and 
experienced personnel perform the procedures  
• following UF guidelines for reducing the risk of COVID-19 
• having available emergency equipment/medications and well-planned 
emergency procedures 
• providing complete confidentiality 
 
Risks associated with COVID-19: Risks related to COVID-19 will be mitigated by strictly 
following UF COVID-19 safety guidelines and the advice of Dr. Cesar Trillo-Alvarez, a 
Co-Investigator and experienced clinician at the UF Health Division of Pulmonary and 
Critical Care. These include: 
• Limiting the number of people in a room and social distancing whenever 
possible. 
• Frequently sanitizing laboratory surfaces and hands.  
• Study personnel wearing KN95 masks and shields, disposable gowns and 
gloves and participants wearing a surgical mask. 
• Upon arrival at the Center for Exercise Science for a scheduled visit, 
participants will be screened for history and symptoms of COVID-19 and body 
temperature will be taken using a contactless forehead thermometer prior to 
exiting their car. If they answer yes to any of the screening questions and/or 
their temperature is greater than 100.4 degrees, then they will need to 
reschedule the visit. Participants will be given a surgical mask and a 2 oz. bottle 
of hand sanitizer. They will be asked to wear the mask and use the hand 
sanitizer prior to entering the building and frequently sanitize their hands during 
the visit and social distance whenever possible.   
• During the pandemic, participants will complete a COVID-19 test 3-5 days prior 
to VO2max testing and only those who are confirmed negative will proceed to 
complete the VO2max test. The COVID-19 test will be prescribed as long as Dr. 
Trillo-Alvarez deems it necessary and will be paid by the study. 
• Exercise sessions will be performed at home and exercise supervision will be 
conducted remotely live via UF Zoom using a wearable device which provides 
real-time heart rate monitoring. 
 
 
 

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Specific risks associated with nitroglycerin: Risks related to nitroglycerin administration 
will be minimized by providing adequate clinical supervision and by taking the following 
safety precautions. An intravenous catheter will be inserted in a peripheral vein prior to 
nitroglycerin administration, in case the effect of nitroglycerin necessitates the 
administration of fluids or medication. ECG will be monitored continuously. Blood 
pressure will be monitored prior to nitroglycerin administration and post-administration 
as frequently as deemed necessary. For individuals whose systolic blood pressure is 
<100 mmHg, nitroglycerin will not be administered. Subjects will remain in bed for 30 
minutes after nitroglycerin administration to allow blood pressure to return to baseline 
before ambulating. Nitroglycerin has been administered safely in our laboratory for more 
than 10 years, and in other laboratories, and clinical situations worldwide.  
 
Risks associated with intra-arterial catheter and cell collection: Risks related to intra-
arterial catheter insertion and cell collection will be minimized by having an experienced 
clinician from the Pulmonary and Critical Care Division, Dr. Cesar Trillo-Alvarez, 
perform the procedure. Dr. Trillo will use ultrasound imaging to guide the cannulation to 
avoid injury to nerves and surrounding structures. Subjects will be screened for 
contraindications including absent pulse at the location of the artery, history of 
Raynaud’s Syndrome, insufficient collateral perfusion based on Allen test, 
coagulopathy, recent use of thrombolytic, anticoagulant or antiplatelet drugs, and partial 
or full thickness burns over the proposed cannulation site. Up to two attempts to insert 
the arterial catheter will be performed; attempts will be aborted if any complications are 
expected. Dr. Trillo will monitor the patient until the arterial line procedure and cell 
collection are completed and no complications are noted.   
 
Specific risks and complications will be mitigated and managed as follows:  
a. Radial Artery Spasm (RAS) 
i. Subjects will be monitored for RAS clinically by physician inserting arterial 
catheter  
ii. Initial management: 
1.  Subjects scheduled to receive nitroglycerin 0.4 mg sublingual (as per 
study protocol to assess endothelium-independent dilation) will 
receive the dose of nitroglycerin as per study protocol and RAS will be 
reevaluated (at 10-min intervals up to 30 min). If no improvement, 
then escalation to higher level of care will be initiated. 
2. Subjects not scheduled to receive nitroglycerin will be monitored for 
severity of RAS. If moderate or severe by clinical judgement, then the 
patient will receive a single dose of nitroglycerin 0.4 mg sublingual 
and RAS will be reevaluated (at 10-min intervals up to 30 min). If no 
improvement, then escalation to higher level of care will be initiated. 
b. Thrombosis 
i. Mitigation of risk:   
1. Cancel procedure if subject has history (or present during evaluation) 
of Raynaud’s phenomenon 
2. Catheter will stay inserted for less than 5 min 
3. No use of large bore catheters, only 20G or smaller 

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4. No insertion if patient has low cardiac output (by history) 
5. No insertion if patient has history of severe peripheral artery disease 
6. No insertion if blood vessel is considered too small for cannulation 
ii. Initial management: If moderate or severe by clinical judgement, then the 
patient will receive a single dose of nitroglycerin 0.4 mg sublingual and RAS 
associated with thrombosis will be reevaluated (at 10-min intervals up to 30 
min). If no improvement, then escalation to higher level of care will be 
initiated. 
c. Dissection, pseudoaneurysm, AV fistula 
i. Mitigation of risk by using procedures described above and 
1. Continuous clinical monitoring at 10-min intervals up to 30 min. 
2. Local pressure to control bleeding and hematoma 
3. If present and no improvement with initial local pressure, then 
escalation to higher level of care will be initiated. 
d. Infection 
i. Mitigation of risk:   
1. All sterile insertion as per usual guidelines. Use of mask, caps, 
gowns, sterile gloves, and chlorhexidine (or equivalent if patient 
allergic) to prepare the skin. 
2. Catheter will be inserted for less than 5 minutes. 
e. Blood loss 
i. Mitigation of risk:   
1. No arterial puncture will be attempted in any subject with systemic 
anticoagulation. 
ii. Initial management: 
1. Bleeding will be controlled by local pressure; monitoring at 5, 10, and 
15 min after removal of arterial catheter. If bleeding persists 
continuous pressure and evaluation at 10-minute intervals up to 30 
min. 
2. If bleeding present and no improvement with initial local pressure, 
then escalation to higher level of care will be initiated. 
f. Neuropathy 
i. Mitigation of risk: 
1. Use of ultrasound guided technique to avoid nerve injury. 
2. Abort procedure if producing pain or neurological injury. 
ii. Initial management: 
1. If severe neurological injury noticed, then escalation to higher level of 
care will be initiated. 
 
Procedure for escalation of care after initial control in research laboratory: 
 
Local initial first aid, including pressure to stop bleeding and vital signs 
monitoring. The patient will be transferred to University of Florida Hospital for 
further evaluation and care if complications arise. If emergent we will contact 911 
for immediate support and transport. We will immediately refer patient to 
University of Florida/Shands Medical Center for any complications for further 
evaluation and therapy. 

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Risks associated with exercise training:  
In general, risks related to exercise training will be minimized by:  
• adhering to the exercise training guidelines published by the American Heart 
Association and the American College of Sports Medicine 
• providing thorough health screening to ensure there are no contraindications for 
exercise training  
• providing remote live supervision of exercise training via Zoom  
 
Minimizing specific risks associated with exercise training: 
Cardiac risks during exercise will be minimized by completing extensive health 
screening tests before subject enrollment to the exercise intervention. Subjects with 
signs of cardiovascular disease or those at elevated risk for cardiac complications will 
be excluded from the study (see exclusion criteria). A physical examination will be 
conducted, and medical history and resting ECG will be reviewed for contraindications 
to exercise. Prior to exercise testing, heart rate and blood pressure will be obtained 
while the subjects rest supine, sitting and standing to screen for cardiac autonomic 
dysfunction. Baseline maximal exercise tests will be performed to rule out the presence 
of silent myocardial ischemia. Because the exercise level during the diagnostic tests will 
be maximal, it should allow identification/exclusion of individuals who are likely to 
develop ischemia during the exercise training. In addition, subjects will be educated on 
how to recognize cardiovascular disease symptoms such as angina, dizziness and 
excessive shortness of breath. They will be asked to inform the research team 
immediately if they experience any of these symptoms during the diagnostic exercise 
test or the exercise sessions. If participants experience a medical emergency during an 
exercise session, we will call 911 while we continue to monitor them via video 
conferencing. In summary, cardiac risks associated with exercise training will be 
minimized, although we recognize they cannot be completely eliminated. Overall, the 
widely accepted benefits of exercising in older adults should outweigh the risks. 
8. Possible Benefits 
The risk-to-benefit ratio is relatively small because the risks of participating in this study are 
relatively small. Subjects may or may not benefit from participating in this research study.  
The subjects will receive information regarding their health at no cost from the extensive 
testing performed for screening purposes (i.e., medical information derived from physical 
examination, diagnostic exercise test, blood pressure measurements, body composition 
analysis including bone density, aerobic fitness, physical activity and dietary analysis, and 
general blood tests including comprehensive metabolic panel, lipid panel, insulin, complete 
blood count with differential).  Although individual responses may vary, exercise training 
has been shown to lead to many health benefits that may improve quality of life or lower 
the risk for heart disease including higher fitness level, improved heart function, healthier 
blood vessels, lower blood pressure, lower body fat and blood lipids, reduced stress and 
depression.  Participation in the proposed studies could also contribute to the advancement 

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of scientific information that may have important implications for exercise prescription for 
preventing cardiovascular disease in aging. The study will use a translational research 
approach to gain mechanistic insight, for the first time, into sex differences in in vivo 
systemic/cellular/molecular adaptations to exercise in older adults.   
9. Conflict of Interest 
There is no conflict of interest to report. 
 

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