Exploring alterations in sleep-related physiological and psychological outcomes across the menstrual cycle in young, healthy women without sleep disorders: a US-based pilot study protocol

Abstract

Introduction

Research suggests that hormonal fluctuations may contribute to sleep-related physiological and psychological outcomes that differentially affect women’s overall health and well-being. Yet, systematic enquiries on this potential interaction across the menstrual cycle are scant.

Methods and analysis

This protocol paper describes a pilot observational study investigating changes in objective and subjective sleep measures, metabolic biomarkers (body temperature, blood glucose and hormonal concentrations) and psychological outcomes (depressive symptoms, menstrual cycle-related pain and psychological distress), in a cohort of healthy premenopausal women aged 18–35, regularly menstruating, and without sleep disorders. Participants’ sleep is monitored every night over the course of two full menstrual cycles using a Food and Drug Administration (FDA)-approved diagnostic ring from SleepImage and via next morning self-reports (ie, sleep diaries). To minimise the likelihood of undiagnosed sleep disorders, participants also complete two nights of at-home polysomnography. Daily hormonal concentrations are assessed via morning urinalysis using the Mira Fertility Monitor while transitions between hormonal phases are further confirmed by biochemical assays. Body temperature, blood glucose concentrations, diet and physical activity behaviours are continuously recorded using wearable devices and smartphone apps from Oura and Levels. The primary outcomes of this study are total sleep time and sleep quality. Secondary outcomes include sleep onset latency, wakefulness after sleep onset, sleep staging, daytime sleepiness, respiratory rate, resting heart rate, heart rate variability and subjective mood. This study will provide novel data to disentangle the intricate relationship between sleep behaviours, mental well-being and menstrual health in premenopausal women.

Ethics and dissemination

The study was approved by the Institutional Review Board at Parker University (protocol number PUIRB-2025-3). Study findings will be presented in peer-reviewed publications and at academic conferences.

Strengths and limitations of this study.

• Changes in sleep-related physiological and psychological metrics are examined via both objective methodologies and subjective measurements over two full menstrual cycles.

• Most biomarkers and variables of interest are continuously monitored throughout the study period.

• Data analysis is stratified by menstrual cycle phase, thus capturing potential changes related to hormonal fluctuations.

• Data collection is conducted at participants’ homes using wearable devices, meaning that compliance and data accuracy may be reduced compared with experimental trials carried out in well-controlled clinical settings.

• Due to its observational nature, this study does not allow us to draw conclusions about causation.

Introduction

Sleep is a universal biological phenomenon essential to many physiological and behavioural processes across the human lifespan.¹ Subjective and objective sleep measures have been associated with variations in metabolism,² endocrine function,² immune function,³ mental and cognitive health,^{4 5} mood,⁵ physical and cognitive performance,⁶,⁹ and disease state in most tissues of the human body.¹⁰ The National Center on Sleep Disorders Research¹¹ recently reported that 30%–40% of adults and 65%–80% of teenagers within the US population present with sleep deficiency. The aetiology of this sleep inadequacy epidemic is the result of numerous factors compromising sleep quantity, sleep quality or both. One contributing factor deserving of particular attention is biological sex—specifically, being female.

Although sleep inadequacies affect both males and females, females tend to report more sleep disturbances and poorer sleep quality than their male counterparts,⁵¹²,¹⁷ with sleep quality decreasing over the course of a woman’s lifespan relative to reproductive status.^{18 19} Given such strong correlation between reproductive status and sleep quality, questions arise on how exactly and to what extent the short-term hormonal fluctuations experienced across the menstrual cycle may affect sleep quality. Indeed, while the precise nature and direction of this relationship is still not fully characterised, emerging evidence suggests that the hormonal fluctuations typical of the female menstrual cycle may induce significant changes to normal sleep-wake patterns.²⁰ Sex differences in sleep quality are so remarkably pronounced that female sex is a known risk factor for sleep disturbances such as insomnia.²¹,²⁵ Notwithstanding, most subjects involved in the available scientific literature surrounding sleep are males,²⁶,²⁸ while the limited data in female subjects largely present mixed results, possibly due to small sample sizes, great heterogeneity in study design and low fidelity measures. When performing research on female cohorts, there are many unique physiological processes to be considered, one of which is the menstrual cycle.

To date, research on sleep-related behaviours in the female population presents conflicting results, so no causal conclusion can be drawn on the effects or directionality of sleep quality, both objectively and subjectively measured, on physiological or psychological markers in relation to the distinct hormonal profiles that characterise the menstrual cycle. Furthermore, most studies evaluate sleep quality based solely on sleep architecture (ie, sleep staging or cycles), which may not accurately or comprehensively capture overall sleep quality in all its dimensions.²⁹ Critical methodological issues are yet to be addressed given that much of prior clinical research does not span the full menstrual cycle, nor does it account for within-subject variability or relevant covariates that may differentially affect sleep, such as nutrition, physical activity and other lifestyle factors. Additionally, most designs use different methods to define menstrual cycle phase, record sleep and analyse objective or subjective sleep data, while not accounting for important autonomic variables known to impact key sleep metrics. Lastly, no studies have provided a reliable, comprehensive and objective measure of sleep quality, that is, a quantitative summation of individual sleep biomarkers known to have a direct relationship with sleep health.³⁰

The purpose of the present study is therefore to analyse the relationship between physiological sleep health with quality metrics and both serum and urine biomarkers across the full menstrual cycle. It is commonly observed that hormonal fluctuations are associated with inadequate sleep and sleep disturbances in times of hormonal transition such as puberty, pregnancy, perimenopause and menopause, yet it remains unclear whether the hormonal fluctuations experienced over the course of a menstrual cycle affect objective variables of sleep in healthy, premenopausal populations. To address this knowledge gap, we designed a novel approach to measure baseline quantitative sleep quality, hormonal biomarkers and fluctuations across two full menstrual cycles in a healthy population of premenopausal women.

Study design and aim

In the following sections, we describe the protocol of our pilot observational study to investigate potential changes in physiological, psychological and sleep-related variables throughout the female menstrual cycle. The distinct hormonal patterns and day-to-day fluctuations characterising different phases of the menstrual cycle have been subjectively associated with several behavioural and physiological outcomes, first and foremost sleep disturbances and mood alterations.^{4 5 18} The primary aim of this study is to identify and investigate such outcomes using a combination of standardised objective measurements, while also accounting for interindividual variations in key metabolic biomarkers. To this purpose, while the number, nature and order of the various tests administered throughout the study is identical for all participants, the testing window and precise timeline of the study is individually tailored to reflect the average length of each subject’s menstrual cycle. This approach to data collection and analysis will allow comparison of different real-life scenarios, with corresponding results applicable to the healthy premenopausal population at large, effectively contributing to the body of knowledge surrounding female health and baseline physiology. Finally, results from this study will be used to inform subsequent enquiries in larger cohorts and hereby corroborate preliminary findings.

Study outcomes

The primary study outcomes are total sleep duration and sleep quality. Both variables are objectively and subjectively assessed throughout the study via nightly recordings using an FDA-approved diagnostic ring, and self-reported evaluations upon waking (sleep diary), respectively. Total sleep duration is measured in minutes, while sleep quality is estimated from two objective measurements: sleep efficiency (ie, the percentage of time spent asleep compared with the total time spent in bed) and multiple autonomic nervous system sleep metrics as a function of the cardiopulmonary coupling (CPC) algorithm.³¹ CPC-based measures will include the validated Sleep Quality Index, sleep stability, fragmentation and periodicity across the night (see Section Objective sleep measurements).

Secondary outcomes include sleep onset latency (SOL, in minutes), wakefulness after sleep onset (WASO, in minutes), time spent in stable/unstable non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep, subjective daytime sleepiness (measured both upon waking and prior to bedtime), nightly respiratory rate, resting heart rate and heart rate variability (HRV). Finally, changes in subjective mood are also explored. These outcomes are assessed every morning and every evening throughout the menstrual cycle against variations in menstrual pain, distal body temperature, blood glucose concentrations, as well as diet and physical activity behaviours.

Safety

The present study is observational in nature and does not include the administration of any exogenous treatment. Self-reported data are gathered by means of validated questionnaires and smartphone applications, while objective measurements are obtained through the continuous use of wearable devices, hormonal self-tracking via urine samples and biochemical assays (ie, blood tests) performed at home by a trained phlebotomist to minimise participant discomfort. The study does not present any safety concerns, and no adverse effects are expected to occur. Regular, over-the-phone check-ins with participants will allow constant monitoring to further ensure that the safety of all testing protocols is maintained.

Ethics and dissemination

This study was approved by the Institutional Review Board at Parker University (protocol number PUIRB-2025-3). Study findings will be presented in peer-reviewed publications and at academic conferences.

Methods

Study setting

Participants involved in this longitudinal, observational study conduct all administered testing in the comfort of their home. They receive the required study equipment in the mail and are provided with clear instruction on how and when to use each component appropriately. Throughout the study period, participants are closely monitored via regular email and telephone check-ins and receive automated daily reminders via text message to maximise study adherence.

Participant eligibility

Eligible participants are young females aged between 18 and 35 years with a healthy body mass index (BMI), no pre-existing health condition and naturally menstruating (average menstrual cycle length between 21 and 35 days). Individuals using any form of oral contraception, hormone replacement therapy, intrauterine devices or presenting with irregular (as indicated by cycle-to-cycle variations exceeding 7 days) or missing periods in the 3 months prior to recruitment are not eligible. Additional exclusion criteria include sleep-related, psychiatric or neurological disorders, history of alcohol or nicotine dependence or abuse, the use of any psychoactive drugs, as well as the use of prescribed or over-the-counter medications known to interfere with normal sleep-wake cycles, mood (eg,sleeping pills, benzodiazepines, anti-depressive medications) or metabolism. Lifestyle factors known to alter common sleep metrics such as the presence of a bed partner, shift work, extended periods of intense physical activity (eg, peak-phase training for elite-level athletes, participation in major sporting competitions) and trans meridian travel across at least two time zones in the month prior to and during the study period further constitute reasons for participant ineligibility. A comprehensive summary of inclusion and exclusion criteria for this study is provided in box 1.

Box 1. Inclusion and exclusion criteria for study participants.

Eligible individuals must fulfil the following criteria:

1. Between 18 and 35 years of age (>3 years postmenarche).

2. BMI ≤30 kg/m².

3. Minimum 7 hours in bed every night.

4. Natural and regular menstrual cycles of average length between 21 and 35 days in the 3 months prior to the study.

5. No history of pregnancy.

6. Provision of signed and dated informed consent form.

7. Stated willingness to comply with all study procedures and availability for the duration of the study.

Individuals who meet any of the following criteria are ineligible to participate in the study:

1. Irregular or missing periods in the 3 months prior to the study.

2. Use of hormonal contraception or hormone replacement therapy in the 3 months prior to the study.

3. Polycystic ovarian syndrome, premenstrual dysphoric disorder or other hormone-related physiological or psychological condition (MEDI-Q≥20 and no diagnosis of PMS or PMDD at screening).

4. Anovulatory cycles (progesterone <16 nmol/L 7–9 days post-LH surge).

5. Reported use of medication known to affect sleep, mood and/or metabolism.

6. Sleep apnoea (AHI>5) or sleep irregularity as confirmed after the first 14 days of sleep recording using an FDA-approved ring-shaped wearable device (see Section Objective sleep measurements).

7. Insomnia or other sleep-related or respiratory disorder, either pre-existing or as determined by a battery of validated questionnaires (ISI≥15, PSQI≥5, ESS≥10 and SHI≥40 at screening) and following two nights of at-home polysomnography.

8. Any medical condition that may produce abnormal electroencephalography (ie, epilepsy, traumatic brain injury or neurological disorder).

9. Clinically relevant cardiovascular abnormalities as evidenced by blood tests performed at the beginning of the study period.

10. Presence of a bed partner.

11. Lifestyle factors such as extended periods of intense physical activity, shift work or trans-meridian travel across at least two time zones within the month prior to the study and/or during the study period.

12. History of major psychiatric disorder, depression or anxiety (DASS-21≥21 at screening).

13. History of alcohol or nicotine dependency or abuse.

14. Use of central nervous system-active or recreational drugs (cannabis, amphetamines, cocaine, antidepressants, opioids, benzodiazepines) in the 3 months prior to the study.

15. Cannot comprehend or speak English fluently.

Participant recruitment

We aim to recruit 29 participants in total, including 4 pilot subjects. Recruitment commenced in March 2025 and is expected to be completed within 12 months, that is, by March 2026. Due to the novelty and exploratory nature of the study, our relatively small sample will serve as a feasibility pilot for measuring primary and secondary outcomes in a larger, subsequent study. Post hoc power calculations using the software G*Power indicated that a participant sample of 29 subjects will allow us to detect statistically significant correlations equal to, or greater than, 0.5 with an 85% power at an alpha level set to 0.05.

Participants are recruited from Parker University through email and social media outreach and directed to an online survey providing extensive information about the aims and design of the study. Participants’ involvement in the study will cease, and their corresponding data will be removed from the final dataset, if subjects violate any of the inclusion or exclusion criteria listed above (see box 1).

Eligibility screening

Participant eligibility assessment is conducted in two stages:

Stage 1: Online and telephone prescreening

Interested individuals provide their contact details and sign the electronic consent form before completing a battery of online questionnaires to assess initial eligibility based on demographic, anthropometric and lifestyle information (ie, age, BMI, menstrual cycle and medical history, sleep hygiene, mental health, trans meridian travel and shift work). They also receive the Participant Information Statement summarising the aims of the study and the research protocol. The research team schedules and conducts a brief telephone interview with potentially suitable individuals to address any queries and confirm study participation.

Stage 2: Final confirmation and data collection

Individuals who agree to participate in the study receive the required study equipment and corresponding instructions via mail. To establish the precise study timeline for each subject, the testing kit provides a QR code to an online survey where participants are asked to confirm receipt of all study equipment and indicate the date when they expect their next menstrual cycle to begin. Testing begins 2 days prior to this expected date, at which point participants also start receiving automated daily reminders via text message.

Every day on waking, participants complete the Karolinska Sleep Diary (KSD) and the self-monitored hormonal tracking via urinalysis (see Section Hormone analysis and menstrual cycle tracking). On waking and before sleep, participants also complete the Karolinska Sleepiness Scale (KSS), the abbreviated Profile of Mood States POMS,³² and a discrete Visual Analogue Scale (VAS) measuring menstrual-related pain (see Sections Screening questionnaires and psychological testing and Mood changes). Additionally, participants complete an initial blood draw to confirm the absence of pre-existing metabolic or medical conditions (see Section Biochemical assays). Finally, participants are instructed to wear a ring-shaped FDA-approved sleep tracking device (see Section Objective sleep measurements) for 14 nights to confirm the regularity of their sleep and wake times and the absence of sleep-disordered breathing. Disqualified participants due to sleep disorders are informed and referred for appropriate medical care. By contrast, if no clinical abnormality is detected at the end of this 14-day period, participants undergo a final screening step involving two nights of at-home polysomnography (PSG) (see Section Objective sleep measurements) to unequivocally confirm the absence of sleep-disordered breathing, detect periodic leg movements and sleep architecture abnormalities, and to establish multiple standard PSG-based sleep statistics that may indicate an undiagnosed sleep problem. Based on the outcome of such comprehensive evaluation, participants either advance into the second phase of data collection (until two full menstrual cycles are completed) or are dismissed. No subjective or objective data collected from ineligible participants during the screening period are retained or included in the study analyses.

Study protocol

Figure 1 presents a schematic representation of the first half of the study protocol (ie, the first menstrual cycle) for two sample participants with an average menstrual cycle of 30 and 26 days, respectively. The distinct hormonal phases of each menstrual cycle are shown (for representational purposes, only), alongside corresponding variations in metabolic and endocrine measures observed in the available literature.

Figure 1. Schematic of cycle 1 with estimated endocrine timepoints and predictive ultradian rhythm patterns⁵⁶ for a 30-day (top panel) and a 26-day menstrual cycle (bottom panel). DBT, distal body temperature; DBTP, distal body temperature peak; EP, oestrogen peak; HRV, heart rate variability; HRVP, HRV peak; LHS, luteinising hormone surge; OV, ovulation; PSG, polysomnography.

Formal testing and data collection commence 2 days prior to the beginning of the next menstrual cycle following recruitment (to account for a potential early start) and continue for two full cycles. In the above example, our hypothetical participants are therefore monitored over a total of 62 and 54 days, respectively.

Several variables of interest are continuously monitored during initial screening and throughout the study period, including participants’ distal body temperature, blood glucose levels, and daily food intake and physical activity. Objective sleep parameters are also continuously recorded every night using a ring-shaped sleep tracking device, while subjective sleep measurements, mental state evaluations and hormonal tracking are conducted in the morning and/or evening by means of validated sleep diaries, sleep and mood questionnaires and urine samples, respectively.

On four occasions over the course of the study period, participants also complete serum and plasma measurements to detect pre-existing metabolic or medical conditions at screening (eg, anaemia) and to confirm expected hormonal profiles at key timepoints throughout the menstrual cycle (eg, successful ovulation, see Section Biochemical assays).

As previously mentioned, throughout the study period, participants receive daily reminders to maximise compliance to the study procedures. The research team further organises email or telephone check-ins if adherence is found to decline at any point during the study.

A summary of all data collection and testing procedures, along with their frequency and scheduled administration throughout the study period, is provided in table 1.

Table 1. Summary and timeline of scheduled assessments throughout the study period.

Measure	Screening	Every morning*	Every evening*	Daily†	Nightly†	Days 15–16 of cycle 1	Cycle(s) day 1-3*	Cycle(s) day+7–9 after LH surge*
Informed consent	✔
Demographic survey	✔
Baseline questionnaires (ISI, PSQI, ESS, SHI, MEDI-Q, PSST, DASS-21)	✔
Sleep diary (KSD)		✔
Sleepiness (KSS)		✔	✔
Mood (POMS)		✔	✔
Menstrual pain (100 mm VAS)			✔
Urinalysis and hormonal tracking		✔
Continuous sleep tracking					✔
Diagnostic sleep testing						✔
Serum and plasma biomarkers							✔	✔
Distal body temperature				✔	✔
Blood glucose				✔	✔
Diet				✔
Physical activity				✔

^*Indicates assessment conducted during both the first and the second menstrual cycles.

^†Indicates continuous recording throughout.

DASS-21, Depression, Anxiety and Stress Scale 21; ESS, Epworth Sleepiness Scale; ISI, Insomnia Severity Index; KSD, Karolinska Sleep Diary; KSS, Karolinska Sleep Scale; LH, luteinising hormone; MEDI-Q, Menstrual Distress Questionnaire; POMS, Profile of Mood States; PSQI, Pittsburgh Sleep Quality Index; PSST, Premenstrual Symptoms Screening Tool; SHI, Sleep Hygiene Index; VAS, Visual Analogue Scale.

Data collection and instruments

Screening questionnaires and psychological testing

Initial eligibility screening is conducted through several validated questionnaires assessing participants’ sleep hygiene, menstrual health and psychological well-being. More specifically, the following self-reported measurements are included: Insomnia Severity Index,³³ Pittsburgh Sleep Quality Index,³⁴ Epworth Sleepiness Scale³⁵ and Sleep Hygiene Index.³⁶ The Menstrual Distress Questionnaire³⁷ is used to provide a representative score of menstruation-related distress, while the Premenstrual Symptoms Screening Tool³⁸ is used to rule out the presence of premenstrual syndrome or premenstrual dysphoric disorder. The Depression, Anxiety and Stress Scale³⁹ is employed to identify potential risks for psychological disorders. The cut-off scores for each of these tools to determine participant eligibility are presented in table 2.

Table 2. Cut-off scores for self-reported screening questionnaires to determine participant eligibility.

Domain	Questionnaire	Scale	Cut-off score
Psychological Well-being	DASS-21	Depression	≥21	Severe-extremely severe
		Anxiety	≥15
		Stress	≥26
Sleep	ISI PSQI	Insomnia Sleep quality	≥15 ≥5	Clinical: moderate-severe Poor sleep quality
	ESS	Sleepiness	≥10	Moderate-severe daytime sleepiness
	SHI	Sleep hygiene	≥40	Extremely poor
Menstrual Health	MEDI-Q PSST	Menstrual distress Premenstrual syndrome or dysphoric disorder	≥20 N/A	Clinically distressful Positive indication

DASS-21, Depression, Anxiety and Stress Scale 21; ESS, Epworth Sleepiness Scale; ISI, Insomnia Severity Index; MEDI-Q, Menstrual Distress Questionnaire; N/A, not available; PSQI, Pittsburgh Sleep Quality Index; PSST, Premenstrual Symptoms Screening Tool; SHI, Sleep Hygiene Index.

Mood changes

On waking and prior to bedtime, participants complete the abbreviated Profile of Mood State^{32 40} questionnaire, a reliable 40-item scale used to assess positive and negative affect on a 5-point verbal frequency scale ranging from 1 (not at all) to 5 (very much).

Subjective sleep measurements

Self-reported sleep measures including sleep duration, sleep quality, SOL and number of awakenings are recorded every morning via completion of the KSD⁴¹ (see table 3). Daytime sleepiness is also assessed both on waking and prior to bedtime using the KSS,⁴² whereby participants rate their current state of alertness on a 9-point scale (1=extremely alert, 9=extremely sleepy).

Table 3. Karolinska sleep diary.

Date:	Hours		Minutes
At what time did you get into bed?
At what time did you attempt sleep?
How long did it take to fall asleep?
Time of final awakening
Time of getting out of bed
How long did you sleep?
Please select the appropriate number
How did you sleep?	1=very poorly	2	3	4	5=very well
Feeling refreshed after awakening	1=very poorly	2	3	4	5=very well
Calm sleep	1=very poorly	2	3	4	5=very well
Slept through	1=very poorly	2	3	4	5=very well
Ease of waking up	1=very poorly	2	3	4	5=very well
Ease of falling asleep	1=very poorly	2	3	4	5=very well
Amount of dreaming	1=very poorly	2	3	4	5=very well

Objective sleep measurements

Objective sleep measurements including total sleep time, sleep quality, sleep-onset latency, WASO, oxygen saturation and time spent in stable/unstable NREM sleep and REM sleep are recorded every night using an FDA-approved CPC-based sleep testing device (SleepImage System). This ring-shaped wearable device extrapolates key sleep metrics from the analysis of coupling and coherence of three distinct signals: HRV, respiratory rate and tidal volume fluctuations. The validity and reliability of CPC techniques in the provision of accurate sleep spectrograms has been extensively described.³¹ The integration of CPC with oximetry further allows the generation of an AHI, which has consistently shown good correlation with conventional, PSG-derived AHI.⁴³ Participants in this study are instructed to wear the diagnostic ring nightly on their preferred finger to generate a comprehensive sleep report.

To confirm participant eligibility in the study and exclude the presence of sleep-disordered breathing, on days 15 and 16 of the study, participants’ sleep is further monitored using an Onera at-home PSG system. This FDA-approved, class IIa medical device consists of four patch-based (wireless) sensors applied to the forehead, chest, abdominal area and lower leg, to capture multiple physiological parameters related to cardiovascular, respiratory and brain functions, including eye and muscle movements, brain and cardiac activity, oxygen saturation, respiratory effort and flow. The accuracy and reliability of the Onera system for establishing the presence of sleep-disordered breathing has been shown to be comparable to attended laboratory PSG testing,^{44 45} hereby providing a practical, and arguably more comfortable testing solution for relevant sleep data collection.

Hormone analysis and menstrual cycle tracking

Hormone analysis and menstrual cycle tracking are performed in the morning via daily urine sample testing using a Mira Hormone Monitor. This tracking device uses lateral flow assays to estimate concentrations of circulating oestrone-3-glucuronide, pregnanediol glucuronide and luteinising hormone (LH) metabolites, which provide an accurate representation of an individual’s hormonal profile within the previous 6–8 hours.⁴⁶ To simplify data analysis and storage, the monitor connects to the corresponding smartphone app and daily test results are available within 21 min.

Morning urinalysis has been shown to correlate strongly with serum hormone tracking.⁴⁷,⁴⁹ Importantly, and contrary to serum measurements, urinalysis is not immediately affected by diurnal pulsatile release, thus providing a composite reading of hormone metabolites over time.⁵⁰ Nonetheless, to further confirm the reliability of this self-administered method in measuring hormonal concentrations and phase transitions, participants are also required to complete two blood tests at key timepoints throughout their menstrual cycle,⁵¹ see Section on Biochemical assays.

Finally, potential distress pre, during or post menstruation is documented every morning and evening by means of a discrete VAS whereby participants are asked to rate the pain they are currently experiencing in relation to their menstrual cycle on a scale from 1 to 10.

Biochemical assays

Over the course of the study period, participants are required to complete a total of four blood tests, as summarised in table 4. These tests are performed at home by a trained phlebotomist. Participants are required to be in a fasted state and to abstain from strenuous physical activity in the 24 hours preceding each test. Biomarkers of interest are outlined in table 5. Given that there is currently no universal consensus on minimum progesterone levels to confirm ovulation, following the recommendations of Janse De Jonge et al,⁵² ovulation will be said to have occurred if serum progesterone exceeded 16 nmol/L in the luteal phase, approximately 7–9 days after the LH surge.

Table 4. Timeline and purpose of required blood tests based on a 30-day menstrual cycle.

Cycle	Blood draw	Purpose	Panel	Cycle day
1	I	Take baseline measurements to exclude participants with potential hyperandrogenism, anaemia, thyroid disorders or metabolic disorders. Confirm follicular phase (ie, low hormone phase).	1	1–3
	II	Confirm ovulation (serum progesterone >16 nmol/L) and general health.	2	23–25
2	III	Confirm follicular phase (ie, low hormone phase) and general health.	2	1–3
	IV	Confirm ovulation (serum progesterone >16 nmol/L) and investigate possible changes from baseline.	1	23–25

Table 5. Biomarkers collected for panels 1 and 2.

Panel 1	Panel 2
Reproductive Health
Oestrone	Oestrone
Oestradiol	Oestradiol
FSH	FSH
LH	LH
Progesterone	Progesterone
Hyperandrogenism
PRL	PRL
Testosterone (total and free)	Testosterone (total and free)
Cortisol	Cortisol
DHEAS	DHEAS
SHBG	SHBG
Anaemia
CBC, including Differential/Platelet	CBC, including differential/platelet
Iron	Iron
TIBC	TIBC
Ferritin	Ferritin
Metabolic Health
HbA1c
CMP 14	N/A
Lipid panel (including total cholesterol to HDL ratio)
Thyroid Health
HbA1c
T3, free and reverse	N/A
T4, free and total
TSH

CBC, complete blood count; CMP, comprehensive metabolic panel; DHEAS, dehydroepiandrosterone sulphate; FSH, follicle stimulating hormone; HbA1c, haemoglobin A1c; HDL, high-density lipoprotein; LH, luteinising hormone; PRL, prolactin; SHBG, sex hormone binding globulin; T3, triiodothyronine; T4, thyroxine; TIBC, total iron binding capacity; TSH, thyroid-stimulating hormone.

Body temperature and heart rate

Distal body temperature and heart rate are continuously monitored using an Oura ring Gen 3 (Ōura Health Oy, Oulu, Finland) to assist with menstrual cycle tracking and capture potential changes that may have a negative effect on participants’ sleep. The Oura ring is a multisensory and water-resistant wearable device combining infrared photoplethysmography with a negative temperature coefficient and a 3-D accelerometer. All sensors are located on the inside of the ring and are in contact with the palm-side of the chosen finger (middle, ring or index finger of the non-dominant hand). Prior to the beginning of the study, participants complete a fitting test to determine their appropriate ring size. They are instructed to wear the ring throughout the study period and to remove it only for charging purposes during dinner time or when showering. Participants synchronise the ring with their phone every morning for the timely provision of their data. The Oura ring has previously been used to measure skin body temperature from the finger in similar research studies.^{53 54} The temperature sensor provides readings every minute and has been found to match research-standard iButton performance⁵⁵ in both laboratory and real-life settings (r²>0.92) by measuring temperature changes as precisely as 0.13°C (0.234°F).

Blood glucose, diet and physical activity

Participants are provided with a Dexcom G7 continuous glucose monitor from Levels to use daily throughout the study. Participants are also required to record their nutrition and exercise habits using the Levels and Oura apps, respectively. These smartphone-based approaches offer a simple and straightforward way to gather detailed information regarding the type, quantity and time of food consumption via manual entries, barcode scan of packaged items or photo captures paired with AI-powered recognition algorithms, hereby encouraging participants’ compliance to routine meal tracking. Similarly, the types, duration and time of daily physical activities are also digitally recorded, either manually or via connection with Apple Health (for iOS users).

Statistical analyses

Statistical analyses will be completed using a commercial statistical package such as SPSS (Release V.30.0.0) or R Studio (V.4.4.2). Descriptive statistics will be presented as means and SD, medians and IQRs, or counts and percentages, as appropriate. We will employ linear mixed models on continuous outcome data to account for repeated measurements, interindividual variability and potential missing datapoints. Spearman correlation analyses between variables of interest will be conducted to disentangle potential associations between subjective and/or objective sleep measurements, lifestyle behaviours, psychological outcomes and hormonal biomarkers across the menstrual cycle. These potential associations will be analysed and stratified by hormonal phase (follicular and luteal) to allow for a dynamic characterisation of the relationship between endocrine fluctuations and sleep behaviour at an individual level. Scores collected at baseline for outcome variables analysed at multiple points throughout participants’ menstrual cycle will be included in the statistical model as covariates. Participants will be coded as random factors, while the menstrual cycle phase and profile (phase: follicular and luteal; profile: low oestrogen and progesterone, high oestrogen and low progesterone, high oestrogen and progesterone) will be coded as fixed factor. The critical p value for statistical significance will be set at 0.05 (two tailed). Effect sizes will be calculated as partial eta squared (ƞ_p²), Cohen’s d and Hedges’ g, as appropriate.

Data management

Any personal or sensitive information obtained for the purpose of this observational study will be treated as confidential and securely stored adhering to the data privacy guidelines of Parker University. All onboarding, consent and questionnaire data will be captured through the Parker University Research Electronic Data Capture System. Participant data will be identified by unique codes, stored with password protection on a secure electronic database and not accessible via the internet. Only designated study investigators will have access to participant data for regular monitoring and analysis.

Patient and public involvement

The design and conduct of this study do not include any patient or public involvement.