The Effects of Menstrual Cycle Phase on Exercise Treadmill Test Results in Premenopausal Women
Conditions: Menstrual Cycle, Exercise Treadmill
Sponsor: Istanbul Medeniyet University

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STUDY PROTOCOL 
 
 
The Effect of Menstrual Cycle on the Results of Treadmill Exercise Test with High Sensitive Cardiac Troponin 
Levels after Exercise in Women 

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Name of Study: 
The Effect of Menstrual Cycle on the Results of Treadmill Exercise Test with High Sensitive Cardiac Troponin 
Levels after Exercise in Women 
 
 
Study Design: 
Observational Study Model: 
Case-Crossover 
Time Perspective: 
Prospective 
Biospecimen Retention: None Retained 
 
Study coordinator and principal investigator: 
Investigator: Mehmet Agirbasli MD [magirbasli] 
Official Title: Medical Doctor, Professor of Cardiology 
Affiliation: Istanbul Medeniyet University, Istanbul Turkey 
Istanbul, Istanbul, Turkey 
 
Co-investigators 
Dr. Aysu Oktay, Affiliation: Istanbul Medeniyet University, Istanbul Turkey 
Istanbul, Istanbul, Turkey 
Supported by: Istanbul Medeniyet University 

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1. OBJECTIVE AND HYPOTHESES 
 
 
Objective: To compare exercise treadmill test (ETT) results in premenopausal women at the early and late 
follicular phases of the menstrual cycle. 
 
 
General Hypothesis: 
False positive results are commonly observed in women after exercise treadmill test (ETT). The studies 
report severe variability in the sensitivity and specificity of ETT in detecting significant coronary artery 
disease (CAD). Sensitivity ranges from 23% to 100% and specificity ranges from 17% to 100% (1, 2). ETT 
utilizes electrocardiography (ECG) along with heart rate (HR) and blood pressure (BP) monitoring during 
exercise, typically involving a treadmill. The major finding of ETT is the ST segment deviation from the 
baseline. False positive stress ECGs are common especially in premenopausal women, limiting the 
specificity of the test (3-6).  
Menstrual periods can cause biological variability in ETT in premenopausal women (7). The menstrual 
period is divided into phases: early follicular (days 1-5), late follicular (days 6-12), ovulation (days 13-15), 
early luteal (days 16-19), mid-luteal (days 20-23) and late luteal phases (24-28 days). Early follicular phase 
is characterized by low estrogen and low progesterone, late follicular phase is characterized by low 
progesterone and high estrogen. Mid-luteal phase is characterized by high progesterone and high estrogen 
levels (7-9). Early and late follicular phases of the menstrual cycle in women demonstrate nearly 4 fold 
differences in estrogen levels. The menstrual period phase affects thermoregulation and physical working 
capacity in women (9, 10). Estrogen has protective and vasodilatory effects on the cardiovascular system 
(11). With the discovery of high sensitive cardiac troponins (hs-cTn), minute changes of hs-cTn have 
become detectable (12). 
The effects of menstrual periodic changes on the results of exercise treadmill test in premenopausal women 
have not been clearly defined. Primary purpose of the study is to investigate the biological causes of false 
positive test results in the treadmill exercise test in premenopausal women. Estrogen is known to have direct 
vasodilatory effects on coronary arteries. Early and late follicular phases of the menstrual cycle are 
characterized by low and high estrogen levels, respectively. The Early Follicular Phase starts on the first day of 
the menstrual cycle and ends when oestradiol begins to increase. It is characterised by increasing LH and 
FSH and constant low levels of oestradiol. The late follicular phase starts with the increase in oestradiol and 
ends at its preovulatory peak (7-10). False positive stress ECGs are commonly reported especially in 
premenopausal women, limiting the specificity of the ETT (3-5). Studies demonstrated that HR adjustment of in 
exercise-induced ST segment depression using the ST/HR slope or ST/HR index could improve the sensitivity and 
specificity of ETT (2, 13-15).  
The hypothesis of the study is that ETT results and hs-cTnT release after ETT will change at the early and late 
follicular phases in premenstrual women.  
 
 

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2. STUDY ENDPOINTS 
 
The major finding of ETT is the ST segment deviation from the baseline. The ST segments are evaluated 60-
80 ms after the J point. ECG positive ETT result was defined as ≥ 1 mm (0.1 mV) horizontal or 
downsloping ST depression in at least 3 consecutive beats.  
 
Chest pain or inadequate hemodynamic response during ETTs are considered clinically positive ETT result. 
Tests that do not meet these criteria but show ST segment changes or had low diagnostic value due to 
baseline ECG changes are considered indeterminate ETT results.  
 
Automated measurements and mathematical calculations are recorded in the ETT system (General 
Electronic GE Healthcare T2100-ST Treadmill & CASE 6.73 Stress Test System).  
 
ETT results are evaluated by 2 different cardiologists and categorized as positive/indeterminate/negative 
ETT results.  
 
Hs-cTn is measured before and 15 minutes after ETT.  
 
Indices of ST/HR slope and the ST/HR index were reported automatically by the General Electronic GE 
Healthcare T2100-ST Treadmill & CASE 6.73 Stress Test System device. The primary endpoint in the study 
was the ST/HR index. The secondary endpoints were ST/HR slope, maxımum ST segment depression (mm), 
HR and BP response to exercise, maximum exercise capacity, and the change in hotmones and hs-cTn after 
ETT. 
 
 
 
Primary endpoint: 
 
ST/HR index during ETTs at the early and late follicular phases of the menstrual cycle  
 
(µV/beats per minute (bpm)) 
Secondary endpoints: 
1. Change in maximal exercise capacity with exercise and menstrual cycle (METs score) 
 
2. Maxımum ST segment depression (mm) 
 
3. ST/HR slope (µV/beats per minute (bpm)) 
 
4. The change in high-sensitive cardiac troponin T after ETT (ng/L) 
 
5. Hormone levels (estrogen) at the early and late follicular phases of the menstrual cycle  
 
 

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3. STUDY DESIGN 
The study is performed in the cardiology clinics of a tertiary care training hospital. The study is approved by the local 
ethics committee (number 2023/0129 on 22/02/2023).  
Study Population: 
 
 
The study population consists of premenopausal women who applied to the outpatient clinic with chest pain 
between February 2023 and July 2023. Patients report that they have a regular menstrual cycle.  
 
Written informed consent is obtained from the patients.  
 
All patients have transthoracic echocardiography. Echocardiography demonstrates normal left ventricular systolic 
and diastolic function. Echocardiography does not show wall motion abnormality or significant valvular heart 
disease.  
 
All patients are evaluated for cardiovascular risk. Risk factors such as hypertension, diabetes mellitus, cigarette 
smoking, hyperlipidemia, family history of CAD, and drug use are questioned (16). Cardiovascular risk factors are 
defined as follows. Hypertension is defined as a persistent elevation in office systolic BP ≥140 and/or diastolic BP 
≥90 mmHg, or a 24-hr ambulatory BP monitor average of ≥130/80, or requirement of chronic use of 
antihypertensives. Hyperlipidemia is defined as elevated total cholesterol (>240 mg/dl), low-density lipoprotein 
cholesterol (LDL-C) (>160 mg /dl), triglycerides > 150 mg/dl, high-density lipoprotein cholesterol (HDL-C) (< 50 mg 
/dl). Cigarette smoker is defined as having smoked at least 100 cigarettes in lifetime. Type 2 diabetes mellitus is 
defined as fasting plasma glucose (FPG) ≥ 126 mg/dL, or 2-hour plasma glucose ≥ 200 mg/dL during a 75-g oral 
glucose tolerance test (OGTT), or random plasma glucose ≥ 200 mg/dL in a patient with classic symptoms of 
hyperglycemia, or hemoglobin A1c (HbA1c) ≥ 6.5% (48 mmol/mol). Self-reported family history of premature CAD 
is defined as having a first-degree relative with premature CAD (men, age <55 years; women, age <65 years). 
 
Menstrual cycle  
The history of the menstrual cycle of the patient is obtained in detail. The duration and regularity of the menstrual 
cycle, the amount of bleeding, and the number of days the bleeding lasted are noted for each patient. Patients with 
irregular menstrual cycles, breakthrough bleeding, spotting, gynecological examination, or treatment plan that 
would disrupt the menstrual cycle are excluded from the study.  
 
The early and late follicular phases are determined from the history. The early follicular phase ETT is planned on 
the 3rd day of menstrual bleeding, while the menstrual bleeding continues. The late follicular phase ETT is planned 
on the 3rd day after the menstrual bleeding ends. Hormone levels are measured before each ETT to ascertain the 
phase of the menstrual cycle. Two ETTs are performed on each participant.  
 
The blood sample is taken 30 minutes before the ETT to measure follicular stimulating hormone (FSH), 
luteinizing hormone (LH), estrogen, progesterone, and hs-cTn. After the test, the patient is allowed to rest for 15 
minutes and blood is taken to check the 2nd hs-cTn after ETT.  
 
 
 

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Sampling Method: Probability Sample 
Minimum Age: 20 years  
Maximum Age: 55 years  
Sex: 
Female  
Gender Based: Yes 
Female patients in the pre-menopausal period 
 
The inclusion criteria are:  
 
1. Premenopausal female patients with regular menstrual cycles,  
 
2. ages between 20-55,  
 
3. Patients have been seen in the cardiology clinic by a cardiologist for the symptoms of chest pain with a 
clinical suspicion of angina or angina equivalent. 
 
4. A cardiologist has ordered ETT as a diagnostic test for the symptoms of chest independent of the study 
protocol  
 
5. Patients can understand and sign the informed consent.  
 
The exclusion criteria are:  
 
1. Women with menopause or menopause-related symptoms or menstrual cycle irregularity or use of 
hormonal therapy or oral contraceptives, current pregnancy 
 
2. patients with a history of CAD,  
 
3. secondary or uncontrolled hypertension,  
 
4. history of valvular heart disease,  
 
5. left ventricular hypertrophy on echocardiography,  
 
6. abnormal systolic or diastolic function or wall motion abnormality on echocardiography, 
 
7. history of cardiac arrhythmia or heart failure, pericarditis or myocarditis 
 
8. history of cancer, stage ≥ 3 kidney failure, liver failure, history of psychiatric illness, history of, recent 
infection. 
 
9. pre-existing ECG abnormality that would confound the interpretation during the ETT (baseline ECG was 
considered abnormal ın the presence of the following findings: prolonged QT interval, chamber 
hypertrophy, alterations in the ST-T waves, any degree of heart block, T-wave abnormalities, premature 
ventricular contractions (PVCs), right bundle branch block (RBBB), left bundle branch block (LBBB), left or 
right axis deviation, or any abnormal rhythm other than normal sinus rhythm).  
 
10. patients who cannot tolerate Bruce protocol in the first ETT  
 
11. lack of ability or mental capacity to understand the study 
 
 
 
 
 
 
 
 
 
 
 

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4. STUDY SCHEDULE 
 
 
 
 
Assessment 
Screening 
visit-(Day 
0) 
Recruitment First ETT 
Phone visit on 
day 7 after 
ETT 
(± 2 days) 
Second 
ETT at a 
different 
menstrual 
phase 
Phone visit 
on day 7 
after ETT 
(± 2 
days) 
Informed Consent Form 
X 
X 
X 
X 
Demographic characteristics 
X 
Blood pressure, heart rate, and other 
vital signs 
X 
X 
X 
X (by 
phone) 
X 
X (by 
phone) 
History/ Physical examination 
X 
X 
X 
X 
Hs-cTn and hormone laboratory workup 
X 
X 
Inclusion/exclusion criteria 
X 
Pregnancy test (serum / urine) 
X 
 
 
 

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5. THE STUDY FLOW is demonstrated below.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 patients who do not meet the 
inclusion/exclusion criteria are 
excluded  
Consequent cases of premenopausal 
women who are referred for ETT are 
screened  
patients who refuse to participate 
in the study are excluded   
premenopausal women who meet 
the inclusion/exclusion criteria are 
offered to participate in the study  
 patients who not come for the 
2nd ETT are excluded  
patients complete first ETT and are 
invited to come for repeat ETT,   
1st and 2nd ETTs are performed at 
different phases of the menstrual 
cycle (early or late follicular phases)  
patients who complete the 1st 
and the 2nd ETTs are included in 
the analysis 

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