The LIFE study: a cross-sectional study protocol for LIfestyle risk Factors for chronic disease across the stagEs of reproductive ageing

Abstract

Introduction

The dynamic physiological and hormonal changes through the menopause transition predispose women to an increased risk of chronic diseases including cardiovascular disease, metabolic disease, depression and dementia. The underlying mechanisms remain unclear, yet it is thought that chronic systemic inflammation and changes to lifestyle behaviours play important roles. The LIfestyle risk Factors for chronic disease across the stagEs of reproductive ageing (LIFE study) is a cross-sectional study aimed to characterise how hormonal and lifestyle (physical activity, diet and sleep) differences across pre, peri and postmenopause influence chronic systemic inflammation, visceral adiposity, cognitive function and sleep health.

Methods and analysis

Women aged between 40 and 65 years were recruited and classified into pre, peri or postmenopausal groups. Body composition measures and blood samples were collected. Sleep and physical activity were objectively measured using activPAL4 and ActiGraph GT9X link accelerometer over 7 days. Participants were also provided with a sleep diary. Physical function was assessed using the Short Physical Performance Battery. Cognitive function was evaluated using Addenbrooke’s Cognitive Examination-III and Cambridge Neuropsychological Test Automated Battery. Participants completed a series of questionnaires: Depression, Anxiety and Stress Scale-21, RuSATED, Berlin Questionnaire, Insomnia Severity Index, Activities-specific Balance Confidence Scale and the Australian Eating Survey.

Ethics and dissemination

Ethical approval was received from the relevant University Human Research Ethics Committee (ethics approval number #S221718) prior to the commencement of the research project. Data collection is ongoing and expected to be completed by April 2026. Results are expected to be available from July 2026. Findings will be disseminated in national and international conferences and in peer-reviewed journals and expected to inform how differences in lifestyle behaviours across menopause influence chronic systemic inflammation, visceral adiposity and cognitive function. Understanding and characterising the links between lifestyle behaviours and menopausal symptoms will inform targeted strategies to improve long-term well-being, heart, brain and metabolic health.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• Comprehensive assessment of behavioural and biological factors, including sleep, physical activity, sedentary behaviour, dietary intake, inflammatory cytokines and body composition, provides a multidimensional understanding of menopausal health.

• Inclusion of multiple lifestyle measures, with device-based assessment where applicable.

• Recruitment of participants from a regional population improves the relevance and translation of findings to non-metropolitan settings.

• The cross-sectional design limits the ability to infer causality or assess changes over time.

• Despite intended adjustment for age, disentangling the independent effects of chronological ageing and reproductive ageing remains challenging in a midlife cohort, and future studies using longitudinal designs and narrower age ranges are needed to minimise residual confounding.

Introduction

The menopause transition is defined as a dynamic period of physiological change, ending in complete ovarian failure and cessation of reproductive function.¹ The rapid decline in the production and secretion of ovarian hormones, particularly oestrogen, is associated with changes in mental health, cognitive, cardiovascular, immune and metabolic functions, and unfavourable changes in body composition.2,5 These changes predispose postmenopausal women to an increased risk of multiple diseases including osteoporosis, hypertension, cardiovascular disease, metabolic disease, depression and dementia.6,9

Lifestyle-related factors, particularly at midlife, play a critical role in the reduction of chronic disease risk and inflammation. Factors such as weight gain, increased sedentary behaviour, poorer sleep quality and diet all contribute to an increase in low-grade inflammation.^{10 11} It is this chronic systemic inflammation that plays an important role in the aetiology of many of the diseases known to increase in prevalence in women after menopause.^{2 6 12 13} Currently, the underlying mechanisms to explain this increase in prevalence in postmenopause and the link to systemic inflammation are poorly understood and little is known about the complex biological changes that occur during the menopausal transition that may contribute to the development of chronic disease.

Incidence of overweight and obesity is increased in postmenopausal women compared with premenopausal women via multifactorial influences including reduced oestrogen production, reduced energy expenditure and lower basal metabolic rate.^{14 15} The withdrawal of oestrogen, specifically 17β-oestradiol, drives a shift in the deposition of adipose tissue from subcutaneous gluteal femoral regions to ectopic sites around metabolically active visceral organs, including the liver, pancreas and skeletal muscle.^{3 13 15 16} As a result, circulating markers of inflammation, glucose and lipids become elevated and instigate disruptions in insulin production, secretion and action, mechanisms which underlie the development of chronic disease.^{4 17 18} These changes to body composition may explain the increase in chronic inflammation in postmenopausal women and subsequent increase in disease prevalence.^{19 20}

Adipose tissue secretes hormones and inflammatory cytokines which play an integral role alongside oestrogen in the regulation of energy intake, expenditure and storage.^{14 21} In overweight and obesity, adipose tissue dysfunction occurs, where an overproduction of hormones and inflammatory cytokines such as leptin, interleukin-1β (IL-1β), IL-6, tumour necrosis factor-a (TNF-α), among others, contributes to a state of low-grade inflammation.22,25

For women approaching menopause, the confounding effect of excess body fat, alongside declining ovarian function and withdrawal of oestrogen, perpetuates chronic inflammation and further heightens the risk of chronic disease.^{26 27} Studies have shown a strong link between increased proinflammatory cytokine activity and risk of cardiovascular disease, metabolic syndrome and type 2 diabetes.^{20 28} Chronic elevations in proinflammatory cytokines are also associated with alterations in cognitive function.^{29 30} The depletion of ovarian hormones during postmenopause has been considered a risk factor for accelerated cognitive decline in women.^{31 32} However, no studies have concurrently investigated the underlying mechanisms between mental health status, cognition, inflammation and increased adiposity that ensue post-menopause.

Very few studies in the literature have compared markers of systemic inflammation across menopause, with significant limitations in the measurement of body composition, classification of menopausal stage and lack of hormone verification of menopause stage.^{2 9 13 33 34} Furthermore, research investigating the relationship between lifestyle factors (eg, diet, physical activity, sleep) and pro-inflammatory markers and the impact on chronic disease progression is sparse. By characterising lifestyle behaviours across menopause, the LIfestyle risk Factors for chronic disease across the stagEs of reproductive ageing (LIFE) study will inform targeted strategies to improve long-term well-being, heart, brain and metabolic health. Understanding the relationship between menopause, inflammation and body composition will direct future lifestyle interventions for older women at risk of obesity-related chronic disease. Finally, investigating the underlying mechanisms linking unfavourable changes in body composition, chronic inflammation, mental health and cognitive function will further our understanding of inflammatory responses to menopause, and how these changes relate to well-being, cognitive decline and increased risk of dementia.

Study aims and hypotheses

The primary aim of the LIFE study is to first, characterise lifestyle behaviours such as sleep, physical activity, dietary intake and pro-inflammatory markers such as leptin, IL-1β, IL-6 and TNF-α between pre, peri and postmenopausal women. The secondary aims are to identify whether the differences/changes in lifestyle factors and pro-inflammatory cytokines in pre, peri and postmenopausal women are associated with visceral adiposity, and to investigate whether differences/changes in pro-inflammatory cytokines and visceral adiposity between pre, peri and postmenopausal women are associated with well-being and cognitive function. The final aim is to investigate the association between lifestyle behaviours (sleep, diet, sedentary behaviour, physical activity) and menopausal symptoms.

It is hypothesised that first, there will be distinct lifestyle behaviours and 24-hour time-use characteristics between menopausal groups. Second, that there will be significant and distinct relationships between inflammation markers and lifestyle measures between each group. Third, that increases in visceral adiposity will correlate with elevated chronic systemic inflammation, and lower well-being scores and cognitive performance in peri and postmenopausal women when compared with premenopausal women. Finally, it is hypothesised that there will be a negative association between lifestyle behaviours and menopausal symptoms across all groups.

Methods

Study design

The LIFE study is a cross-sectional study conducted at the host institution. Recruitment commenced in October 2022. Data collection is ongoing and is expected to be completed by April 2026. Results are expected to be available from July 2026. Ethical approval was received from the University of the Sunshine Coast Human Research Ethics Committee (HREC) (ethics approval number #S221718) prior to the commencement of the research project. Participants provide written informed consent, are screened for menopausal status in the first assessment session and undergo two subsequent assessment sessions to collect demographic and lifestyle data.

Patient and public involvement

Patients or the public WERE NOT involved in the design, conduct, reporting or dissemination plans of the research.

Sample size calculation

A sample size estimate was calculated using G*Power (V.3.1.9.4). As the primary outcomes of the LIFE study are to characterise lifestyle behaviours and investigate associations between inflammatory markers across pre, peri and postmenopausal groups, an effect size of 0.30 was pooled from previous published studies investigating differences in IL-6 concentrations between pre and postmenopausal women.^{3 35 36} Using an alpha level of 0.05 and a power level of 95%, it was determined that a total sample size of n=175 was required for analysis of covariance analysis of three groups and three covariates.

Participants and study setting

Participants are eligible if they identify as a cisgendered woman or non-binary (female sex at birth and gender identity is non-binary) (online supplemental table 1), are aged between 40 and 65 years, have a body mass index (BMI) of 18.5–39.9 kg/m², are able and willing to provide informed consent and live locally to or are willing to travel to the study location. Participants are excluded if they were pregnant or breastfeeding, using exogenous hormones such as hormonal replacement therapy or oral contraceptives, have undergone treatment or surgically induced menopause (eg, oophorectomy, hysterectomy) or have a diagnosis of a hormonal or ovarian disorder such as polycystic ovarian syndrome or hypothalamic amenorrhea. To ensure an equal distribution of participants across the three groups (pre, peri and postmenopausal), once the target number of 58 participants is achieved in each group, recruitment for that group ceases. Complete details of eligibility and exclusion criteria are provided in online supplemental table 2.

Recruitment sources

Recruitment is undertaken using a multimodal strategy to maximise reach across the local community and surrounding regions. Study advertisements are disseminated across the host institution campus, including digital newsletters, internal mailing lists and physical noticeboards. Additional recruitment methods include distribution of study materials through women’s health clinics, and hospitals within the local community, online social media platforms, television and radio feature segments, community announcement boards, local community groups and participant word-of-mouth. All recruitment materials direct interested individuals to the LIFE study-specific webpage hosted by the institution.

Screening and consent

Interested participants self-refer by submitting their details, including name, contact information, residential postcode, date of birth and self-classified menopause status, via an online form located on the LIFE study-specific webpage through the host institution. On receipt of an expression of interest, researchers provide participants with an information consent form via email, along with a link to an online screening form to assess eligibility. Eligible participants are contacted to discuss study procedures through a telephone-based interview and invited to participate in the study. Once participants agree to participate, they are scheduled for two in-person assessment sessions, conducted 1 week apart. A confirmation email is sent with details of both assessment sessions, and a map to the testing location. Eligible participants provide written, informed consent at their first visit, by signing the Participant Information and Consent Form, which is witnessed and signed by a study team member. Ineligible participants are notified and referred to alternate suitable research studies when possible. Participants who cannot be reached are followed up with repeated contact attempts (up to three attempts). The flow of participants through the study, including reasons for exclusion to date, are shown in figure 1.

Figure 1. Participant flow diagram. Interested participants submitted their details via an online form located on the LIFE study-specific webpage through the host institution. On receiving an expression of interest, researchers provided participants with an information consent form via email, along with a link to an online screening form to assess eligibility and menopausal status. Allocation of eligible participants, number of dropouts and reason are shown above. LIFE, LIfestyle risk Factors for chronic disease across the stagEs of reproductive ageing.

Study protocol

First data collection: menopausal classification

2–3 days prior to the initial in-person assessment, a reminder email is sent to participants to confirm the date, time and pretesting preparation. They are asked to complete the Reproductive Ageing in Women (RAW) questionnaire³⁷ via the online platform Qualtrics. Based on their responses, participants are classified into pre, peri or postmenopausal groups. Participants with a regular menstrual cycle mostly the same length apart, are classified as premenopausal. Participants are classified as perimenopausal if they have a variable menstrual cycle (defined as a difference of 7 or more days in cycle length between cycles). Participants who have not experienced a menstrual cycle for 12 months or more due to physiological menopause are classified as postmenopausal. In addition, participants are asked whether they have noticed any changes in menstrual bleeding including heaviness of flow and/or changes in menstrual pain (increase or decrease). They are also asked if they are currently taking or using medications, supplements (including vitamins) or any alternative therapies (eg, acupuncture or yoga) to manage menopausal related symptoms. The RAW questionnaire takes approximately 30 min to complete and includes 19 items related to menstrual cycle length and characteristics, hormonal contraception use, time since the final menstrual period (for postmenopausal participants), cause of menstrual cycle cessation and use of menopausal treatments. The following questionnaires are also completed alongside the RAW questionnaire: the Menopause-specific Quality of Life Questionnaire (MENQOL),³⁸ Alcohol Use Disorders Identification Test - Consumption (AUDIT-C),³⁹ Active Australia Survey,⁴⁰ sitting time questionnaire⁴¹ and the Pittsburgh Sleep Quality Index (PSQI).⁴²

Second data collection: Demographics, blood sampling and lifestyle measures

Participants arrive at the study location campus between 05:00 and 09:00 wearing enclosed shoes and light, non-restrictive clothing with no zips, underwires or metal and having fasted (≥8 hours). After providing written consent, participants complete a brief questionnaire collecting contact details, next of kin and local general practitioner (GP) information, medication use and medical history. Medical history includes details regarding prior radiation exposure to assess whether participants are permitted to undergo bone mineral density (BMD) and body composition scans.

Demographic and lifestyle measures are then collected. Measurements include body mass, standing height, waist and hip circumferences and assessments of BMD and body composition using dual X-ray absorptiometry (DXA), bioelectrical impedance spectroscopy (BIS) and air displacement plethysmography (ADP). Participants are then fitted with an activPAL4 device (worn on the thigh), and an ActiGraph GT9X Link watch (worn on their non-dominant wrist) to monitor physical activity and sleep objectively over a period of 7 days. Participants are also provided with a sleep diary and instructed how to complete it. Blood glucose is measured via capillary whole blood collected from the medial or lateral aspect of the third, fourth or fifth finger using a sterile lancet device. Capillary blood is then analysed instantly (Accu Chek Instant S Meter Kit) to determine fasting blood glucose levels. Blood sample collection for pre and perimenopausal women with regular menstrual cycles is scheduled between days 2 and 7 of their menstrual cycle.⁴³ Perimenopausal women who have not experienced a regular cycle in the 3 months prior, and postmenopausal women are scheduled on a day that is most convenient for the participant.

Third data collection: Physical and cognitive assessments

The third data collection session takes place face-to-face at the study location campus, at least 6 days after the first assessment (average 7.6±2.0 days, range: 6–23 days) in a non-fasted state, with participants refraining from caffeine or alcohol ≥4 hours and strenuous exercise ≥12 hours before arrival. Participants return the sleep diary and monitoring devices, after which seated blood pressure is measured. Physical function is assessed using grip strength, 30-second sit-to-stand, balance tests (semi-tandem, tandem, single-leg), timed-up-and-go, and a 6-minute walk test. Cognitive function is assessed with the Addenbrooke’s Cognitive Examination (ACE-III),⁴⁴ and the Cambridge Neuropsychological Test Automated Battery (CANTAB).⁴⁵ Participants also complete a series of validated questionnaires including the Depression, Anxiety and Stress Scale-21 items (DASS-21),⁴⁶ RuSATED,⁴⁷ Berlin Questionnaire,⁴⁸ Insomnia Severity Index (ISI),⁴⁹ Pre-Sleep Arousal Scale (PSAS),⁵⁰ Activities-specific Balance Confidence Scale (ABC)⁵¹ and the Australian Eating Survey (AES). The study protocol is detailed in figure 2.

Figure 2. Flow diagram of LIFE study protocol. ABC, Activities-Specific Balance Confidence scale; ACE-III, Addenbrooke’s Cognitive Examination-III; ADP, air displacement plethysmography; BIS, bioelectrical impedance spectroscopy; CANTAB, Cambridge Automated Neuropsychological Test Automated Battery; DASS-21, Depression Anxiety Stress Scale; DXA, dual X-ray absorptiometry; ISI, Insomnia Severity Index; LIFE, LIfestyle risk Factors for chronic disease across the stagEs of reproductive ageing; MENQOL, Menopause-specific Quality of Life Questionnaire; PSAS, Pre-Sleep Arousal Scale; PSQI, Pittsburgh Sleep Quality Index; RAW, Reproductive Ageing in Women questionnaire.

Data collection

Data collection procedures used across the three assessment sessions are detailed below. Online supplemental table 3 provides a summary of the objective and questionnaire-based measures along with their intended purpose in the study (eg, primary outcomes, secondary outcome).

Classification of menopause status and symptoms

The RAW questionnaire contains questions to assist in classifying the participants’ menopause status according to the Stages of Reproductive Ageing Workshop (STRAW+10) criteria.⁵² To classify menopause status, participants are asked questions such as current menstrual cycle status and regularity of their menstrual cycle. Length of time spent with menstrual cycle variability or time since the final menstrual period, and cause of menstrual cycle cessation is also reported. In addition, participants are asked whether they have noticed any changes in menstrual bleeding including heaviness of flow, and/or changes in menstrual pain (increase or decrease). Hormonal contraceptive use, hormonal therapy and associated symptoms are recorded. This questionnaire was developed for use in the LIFE study, and its measurement properties are currently being evaluated for reliability and validity.³⁷

As part of the RAW questionnaire, participants complete the MENQOL, which is self-administered and consists of a total of 29 items in a Likert-scale format. Participants respond to questions in each of the four domains of menopausal symptoms, as experienced over the last month: vasomotor (items 1–3), psychosocial (items 4–10), physical (items 11–26) and sexual (items 27–29). Participants rate each symptom as present or absent. If present, its level of bothersomeness was scored on a 0 (not bothersome) to 6 (extremely bothersome) scale. Means will be computed for each subscale by dividing the sum of the domain’s items by the number of items within that domain. Non-endorsement of an item is scored a ‘1’ and endorsement a ‘2’, plus the number of the rating, so that the possible score on any item ranges from 1 to 8.³⁸

Anthropometry measures

Body mass is measured to the nearest 0.1 kg using a set of electronic stand-on scales (AND HW-200GL, A&D Australasia Pty Ltd, Thebarton, South Australia) where participants are asked to wear comfortable light clothing, empty their pockets and remove footwear. Height is measured to the nearest 0.1 cm using a wall mounted stadiometer with the participants’ head positioned in the Frankfort plane, feet together with their back against the stadiometer. Both measures of weight and height are undertaken in accordance with the International Society for the Advance of Kinathropometry protocols.⁵³ BMI is calculated as weight (kg) divided by the square of height (m²).⁵⁴

Waist and hip circumference: Participants stand in an upright position while waist and hip measurements are conducted. The tape is passed around the; waist: abdomen at the narrowest point between the lower costal and top of the iliac crest; hip: widest point of the gluteals/hips. Measurements are completed in duplicate with the mean value recorded as per previously published protocols.⁵⁵ Waist-to-hip ratio is calculated as waist circumference (cm) divided by hip circumference (cm).

Assessment of body composition and bone mineral density

Body composition measures: DXA is used to determine body composition (fat mass, lean body mass, body fat percentage, android and gynoid fat mass and android/gynoid ratio) and BMD. The DXA system is calibrated with phantoms as per the manufacturers’ guidelines each day before measurements are taken. Participants are positioned in the centre of the densitometer table with their hips and knees flexed on a supportive block to flatten the lordosis of the lumbar spine. Antero-posterior spine BMD measurements are obtained using the L1–L4 vertebral bodies. For left hip BMD, the measurement is performed after aligning the long axis of the femur with the scanner. A positioning device is used to internally rotate the femur to elongate the femoral neck. BMD measurements are obtained using the femoral neck, greater trochanter, Ward’s area, intertrochanteric regions and total hip. Participants are re-positioned to the centre of the densitometer table for the total body scan⁵⁶ with their feet placed in a radio-opaque Styrofoam block, secured using a Velcro strap around the ankles to maintain a distance of 15 cm between the feet.⁵⁷ Participants position their hands laterally against the hips within shaped Styrofoam blocks with a consistent gap of 3 cm between the palms and trunk. The arms are held in place with a Velcro strap to minimise any subject movement during the scan.⁵⁷ This positioning protocol has previously been shown to enhance measurement precision, and minimise typical errors associated with technical and biological variability of DXA measurements of whole and regional body composition when compared against the traditional National Health and Nutrition Examination Survey (NHANES) protocol.⁵⁷ All DXA scans are undertaken using the GE Lunar iDXA (Lunar DPX, GE Healthcare, Madison, Wisconsin, USA) and analysed using software (GE encore V.18.0) provided by the manufacturer (GE Healthcare) and according to the manufacturer’s instructions. All scans are conducted by DXA trained and accredited research personnel at the host institution. Participants wear exercise clothing and are instructed to remove shoes, hats and any metal-containing items such as belts, watches, jewellery or coins.

Total body water measurements: BIS (Imp SFB7) is used to assess total body water. The BIS is calibrated as per manufacturers’ guidelines each day before measurements are taken. Participants are supine for a minimum of 10 min prior to, and for the duration of the assessment. Two single tab electrodes (3M Healthcare) are placed on the left hand, next to the wrist joint and on the dorsal surface of the hand proximal to the middle knuckle with at least a 5 cm gap between them. Two electrodes are placed on the left ankle, on the dorsal surface of the foot at the level of the protruding bone on the side of the ankle, and the dorsal surface of the foot with at least a 5 cm gap between them.

Body mass and volume measures: ADP technique using the BOD POD (Cosmed, Italy) system is used to measure the participant’s mass and volume via electronic scale and air displacement to derive body density. The BOD POD system is calibrated as per manufacturer’s instructions each day before measurements are taken. Participant mass is measured, and the participant is then required to sit in the chamber for approximately 5 min. Thoracic gas volume is predicted. As per operation guidelines, the average of two scans is taken as the result for one assessment. Unless the variance is greater than 150 mL, then a third measure is taken, and the mean used for subsequent analysis. Participants wear exercise clothing and a swimming cap to minimise measurement error.

Device-based measurement of physical activity, sedentary behaviour and sleep

Device-based measures of physical activity and sleep are collected using the activPAL4 activity monitor (PAL Technologies Limited, Glasgow, UK; 24 hours protocol) worn on the thigh and the ActiGraph GT9X Link (ActiGraph Corporation, Pensacola, Florida, USA) worn on the wrist of the non-dominant hand. The devices are worn continuously for 7 consecutive days and are removed only for bathing and water-based activities (eg, swimming).

The activPAL4 is initialised and waterproofed with a plastic sleeve and attached to the participants approximately one-third from the top of the thigh using either standard (3M Tegaderm) or hypoallergenic (Opsite Flexifix Gentle) film. Data are imported and analysed using the PALanalysis V.8.11.8.75, CREA Algorithm V.1.3. Data are deemed valid if at least one full day of recording is available (≥10 hours wear time). The activPAL4 uses triaxial accelerometry to reliably discriminate periods of upright activity from seated or lying activities.⁵⁸ The following outcomes will be derived from the ActivPAL4 data: standing time, stepping time, light physical activity (cadence ≥75, duration >1 min), moderate to vigorous physical activity (cadence ≥100, duration >1 min),⁵⁹ overall sitting time (mins), overall prolonged sitting time (≥30 min) and activity score (Metabolic Equivalent of Task Hours; MET.h).

The ActiGraph GT9X Link tri-axial accelerometer is initialised with a sampling rate of 90 Hz and is set to begin recording at midnight following the day the participants receive the device. ActiGraph data are downloaded using ActiLife software (V.6.13.5) using a 60 s epoch and a minimum wear-time of 4 days, with the inclusion of at least 1 weekend/non/workday as this has been shown to be a valid and reliable estimate of weekly sedentary behaviour and physical activity level.⁶⁰ Accelerometer data is processed by removing non-wear periods, trimming the start and end of recording, and checking for missing values in accordance with established guidelines.⁶¹ ActiGraph accelerometer physical activity data are processed using existing validated criteria and procedures.⁶² The Cole-Kripke algorithm⁶³ in ActiLife examines sleep data (Total sleep time, sleep efficiency, sleep timing, regularity). Sleep periods are manually cross-referenced against participant sleep diaries and by visual inspection. This is conducted in line with the Society of Behavioural Sleep Medicine accelerometry guidelines.⁶¹ Data are omitted at the beginning and end of the recording when the device is not on the patient’s wrist. Any non-sleep periods detected as sleep (ie, when the device is taken off the wrist and not worn for a period of time) are removed and validated against the activity monitor logs and sleep diary. ‘In bed (or lights out)’ and ‘out bed (wake up, time stopped trying to sleep)’ times are compared between the ActiGraph with the sleep diary and adjusted where necessary, that is, if the participant in the sleep log indicates they were awake, but lay in bed for another hour, this is adjusted on the ActiGraph data to reflect ‘out bed’ time as time woke up in the sleep diary. Instructions and an activity log are given for both devices where participants note each time the device is removed for bathing or water activities.

Venous blood sample collection

Capillary whole blood is collected using a sterile lancet from the medial or lateral aspect of the third, fourth or fifth finger. Capillary blood is then analysed instantly (AccuChek Instant S Meter Kit) to determine fasting blood glucose levels. Venous blood samples (20–30 mL) are then collected from the antecubital vein using a 21-gauge butterfly needle into prepared vacutainers (PAXgene, serum, and EDTA plasma) by a qualified phlebotomist. Samples are collected at a single time-point in the morning in a fasted state and stored on ice until preparation. After collection, serum samples are allowed to clot for 20–30 min at room temperature. Once serum is clotted, plasma and serum samples are centrifuged at 3000 rpm for 10 min at 4°C. Separated serum and plasma are aliquoted, boxed and frozen in a –80°C freezer before analysis. Blood collected in PAXgene tubes is stored in a −20°C freezer before analysis.

Blood pressure

An automatic digital blood pressure (BP) (OMRON) machine is used to assess BP. Participants are required to sit quietly for a minimum of 5 min prior to the measurement.

Inflammatory, neurotrophic and bone factors

Markers of inflammation (leptin, adiponectin, TNF-α, IL-1β, IL-6, IL-8, IL-10, interferon-γ, monocyte chemoattractant protein-1), bone turnover (osteopontin and osteoprotegerin) and neurotrophic factors (brain-derived neurotrophic factor and vascular endothelial growth factor A), are ascertained from serum/plasma samples collected at assessment session two. Samples are analysed using multiplex microsphere-based immunoassays (Luminex, Thermo Fisher Scientific, or similar).

DNA is extracted using automated column-based techniques (Qiagen) from PAXgene tube samples and used for whole genome sequencing, genome-wide association studies, single nucleotide polymorphism analysis for growth-related factors, DNA methylation studies and RNA expression studies.

Hormonal factors

Markers of reproductive function (oestradiol and follicle stimulating hormone) are ascertained from blood serum collected from participants in assessment session two. Samples are analysed using commercially available enzyme-linked immunoassays (Invitrogen). All blood biomarker analysis (figure 3) is conducted in the Molecular Biology laboratory at the host institution.

Figure 3. Blood-based biomarker assays to be completed under the LIFE protocol. Blood sample collection took place during the second data collection session and all blood biomarker analysis was conducted in the Molecular Biology laboratory at the host institution. These included markers of inflammation (TNF-α, IL-1β, IL-6, IL-8, IL-10, IFN-γ and MCP-1), markers of bone health (OPN and OPG), markers of cognitive function (BDNF and VEGF-A) and finally markers of reproductive function (E2 and FSH). APOE, apolipoprotein E; BDNF, brain-derived neurotrophic factor; E2, oestradiol; FSH, follicle stimulating hormone; IFN-γ, interferon-γ; IL, interleukin; LIFE, LIfestyle risk Factors for chronic disease across the stagEs of reproductive ageing; MCP-1, monocyte chemoattractant protein-1; OPG, osteoprotegerin; OPN, osteopontin; TNF-α, tumour necrosis factor-α; VEGF-A, vascular endothelial factor-A.

Physical performance

Dominant and non-dominant hand grip strength is assessed using a spring-loaded grip dynamometer (TTM, Tokyo, Japan) to estimate physical performance and muscular strength.⁶⁴ Participants perform a maximal contraction with alternating hands maintaining a 90-degree elbow flexion and limiting accessory movements. A brief rest period (approximately 10 s) is provided between subsequent attempts.⁶⁴ The average of three measurements is taken as one assessment.

The 30-second sit to stand protocol is used to assess functional leg strength. Participants are asked to sit in a hard-backed chair, with a seat height of 43 cm from the floor, with their arms folded across their chest. Participants are instructed to rise as fast as possible to a full standing position then return to a full sitting position without using their arms.⁶⁵ The assessor uses a standardised script and provides balance support if necessary.

Balance is measured over three postures, tandem, semi-tandem and single leg balance. Participants are provided with an explanation and demonstration of each position and asked to balance each progressive position for 30 s with their eyes open. Participants are provided with two attempts at each position and the best time at each stage is recorded to the nearest 0.1 s.⁶⁶ Participants are provided balance support until stable, after which timing commences.

The timed up and go test is a validated measure used to assess walking ability and mobility in older adults.⁶⁷ Starting in a seated position, participants are instructed to rise on the researcher’s command ‘Go’, walk forward (marked), turn around, walk back to the chair and sit down. Using a stopwatch, the research staff measure the time taken from their instruction to stand until the participant sits down again.

The 6-minute walk test is a practical measure frequently used in clinical settings as a submaximal test to assess functional exercise capacity.⁶⁸ A 30 m track is marked in an undercovered area outdoors. Using a stopwatch, the research staff instruct the participant to walk as far as they can in 6 min, with the number of laps completed and time recorded by the researcher. The total distance travelled during this time is then calculated.

Neuropsychological testing

The ACE-III is a brief, paper-based cognitive test that assesses multiple domains of cognitive function including attention, memory, fluency, language and visuospatial abilities.⁴⁴ A trained member of the research team asks the participant a series of set questions and instructions in a quiet room. The test takes approximately 15 min to complete. The neuropsychological testing and corresponding cognitive domains are detailed in figure 4.

Figure 4. Neuropsychological testing and corresponding cognitive domains. Cognitive function assessment took place in the third data collection session. Global cognitive function was assessed with the ACE-III. Cambridge Automated Neuropsychological Test Assessment Battery was used to assess the cognitive domains of executive function, attention and psychomotor speed and memory.

The CANTAB⁶⁹ is a computerised battery of cognitive assessments used to evaluate a range of cognitive functions including attention, psychomotor speed, executive function and memory. CANTAB is self-administered using an iPad touch screen interface and takes approximately 50 min to complete. Participants are asked to follow the audio instructions played through the application to perform the motor screening task (MOT), which is used to introduce the CANTAB touchscreen to participants. The MOT provides a general assay that tests whether sensorimotor or other difficulties limit the collection of valid data from each participant. After touchscreen adaptation, the following CANTAB tests are completed, paired associates learning, spatial working memory, delayed matching to sample, verbal recognition memory and reaction time. CANTAB outcomes are automatically recorded in the application for later retrieval by study staff. All access to the application is controlled by the Licensee and access is granted via a password-controlled interface.

Questionnaire measures

Questionnaires are administered during the testing session or emailed to the participants for self-completion. The RAW, MENQOL,³⁸ Active Australia,⁴¹ PSQI,⁴² AUDIT-C³⁹ and family disease history questionnaire are combined and completed in assessment session one online. The remaining questionnaires (DASS-21, ABC, Berlin Questionnaire, RUSATED, ISI, PSAS) and chronotype preference are administered together during assessment session three.

The DASS is a 21-item self-report instrument designed to measure the presence and severity of three related negative emotional states—depression, anxiety and stress—over the past 7 days.⁷⁰ Participants are asked to respond to each item on a scale ranging from 0 to 3 (ie, 0 being ‘did not apply to me at all’ and 3 being ‘applied to me very much, or most of the time’). The DASS is clinically validated in measuring emotional states of depression, anxiety and stress in clinical populations.^{46 71}

To assess the falls risk of each participant, the ABC scale is completed at all testing time points.⁵¹ The ABC scale is a 16-item scale that requires participants to answer questions relating to their balance confidence when performing activities at home or in areas external to home. Each item is rated from 0% (no confidence) to 100% (complete confidence). The answers are summed and then divided by 16 to obtain a total ABC score. The ABC scale has been shown to have strong test–retest reliability, internal consistency and validity.⁵¹

Self-reported physical activity, sitting time and sleep

The Active Australia Questionnaire is used to measure the number of sessions and time spent per week engaged in walking, vigorous gardening/yard work and vigorous physical activity (eg, recreational sports, weight training). Participation in physical activity, measured as number of sessions and time per week for each question is calculated.⁴¹ Sitting time is assessed using the sitting time questionnaire. Participants report their sitting time over the week prior, categorised by weekdays and weekends, and across five contexts: work, transport, television viewing, leisure time computer use and ‘other’ sitting.

A sleep diary is provided to participants to be completed over 7 consecutive days, to track sleep patterns, monitor regularity and inform an accurate assessment of actigraphy recordings.⁷² The sleep diary is based on the Consensus sleep diary and assesses various aspects of sleep, including sleep and wake time, sleep duration and napping.⁷² The questions ask about: (1) the time of getting into bed; (2) the time at which the individual attempted to fall asleep; (3) sleep onset latency; (4) number of awakenings; (5) duration of awakenings; (6) time of final awakening; (7) final rise time; (8) perceived sleep quality (rated via Likert scale); and (9) an additional space for open-ended comments. An example of the sleep diary is given in online supplemental table 4.

The 24-item PSQI assesses subjective sleep quality and disturbances in the preceding month. Five items, intended for a bed partner or roommate provided descriptive value only. The other 19 items are divided into seven ‘component’ scores (subjective sleep quality, sleep latency, duration, efficiency and disturbance, use of sleep medication and daytime dysfunction), each of which has a range of 0–3 points. In all cases, a score of ‘0’ indicates no difficulty, while a score of ‘3’ indicates severe difficulty. The seven-component scores are then added to yield a ‘global’ score, with a range of 0–21 points, ‘0’ indicating no difficulty and ‘21’ indicating severe difficulties.⁴²

The ISI is a seven-item measure of the nature, severity and impact of current (ie, past 2 weeks) insomnia symptoms, with participants responding on a Likert-type scale (ie, 0 being ‘not at all’ to 4 being ‘very much’). The total score ranges from 0 to 28, with a higher score indicating more severe insomnia.⁷³ The questions relate to the subjective qualities of the respondent’s sleep, including satisfaction with sleep patterns, the degree to which insomnia interferes with daily functioning and whether symptoms are noticeable to others.⁷³

RUSATED is a self-administered questionnaire evaluating six key dimensions of sleep health shown to be associated with various health outcomes: (1) regularity (‘do you go to bed and get out at the same times (within one hour) every day?’, (2) satisfaction (‘are you satisfied with your sleep?’), (3) alertness (‘do you stay awake all day without dozing?’), (4) timing (‘are you asleep (or in bed) between 2.00am and 4.00am?’), (5) efficiency (‘do you spend less than 30 minutes awake at night?’) and (6) duration (‘do you sleep between 6 and 8 hours per day’). Each item is rated on the frequency of meeting the criteria for each dimension from 0 to 2, with 0 for ‘never’ or ‘rarely’, 1 for ‘sometimes’ and 2 for ‘usually’ or ‘always’.⁷⁴

The Berlin Questionnaire (BQ) assesses the degree of risk for obstructive sleep apnoea, incorporating questions about snoring (category 1), daytime somnolence (category 2) and hypertension and BMI (category 3). The overall BQ score is determined from the responses to the three categories: scores from the first and second categories were positive if the responses indicated frequent symptoms (>3–4 times per week), whereas the score from the third category was positive if there was a history of hypertension or a BMI >30 kg/m². Patients are scored as being at high-risk for obstructive sleep apnoea if they had a positive score on two or more categories, while those who did not were scored as being at low-risk.⁴⁸

The PSAS is a 16-item self-report questionnaire designed to assess arousal levels before falling asleep (ref). It consists of two subscales, these being cognitive and somatic, both demonstrating adequate internal consistency (α=0.82 and 0.79, respectively).⁵⁰ The PSAS is rated on a 5-point Likert scale ranging from 1 (‘not at all’) to 5 (‘extremely’) with total scores for each subscale ranging from 8 to 40. Higher scores indicate greater pre-sleep arousal.⁵⁰

The self-morningness/eveningness is a single item scale designed to assess chronotype preference, ‘How would you describe yourself’ (ie, (1) definitely a morning person, (2) more a morning than an evening person, (3) both morning and evening person, (4) more an evening than a morning person, (5) definitely an evening person, (6) do not know), which has previously validated.^{75 76}

Dietary intake

Caffeine use is determined through a single question asking ‘Thinking about caffeinated beverages such as energy drinks, soft drinks, coffee and tea, how many cups or cans of caffeinated beverages do you typically drink each day?’ Responses ranged from 1 to 10 or more or refuse to answer. Habitual dietary intake is assessed using the Australian Eating Survey (AES), a validated 120-item semi-quantitative food frequency questionnaire⁷⁷ with 15 supplementary questions regarding age, use of vitamin supplements, food behaviours and sedentary behaviours. The AES is a self-administered online tool assessing dietary intake over the preceding 6 months. The tool is administered using an iPad during assessment session three. An individual response for each food, or food type, is required, with frequency options for food ranging from ‘never’ to ‘4 or more times per day’, dependent on the food and up to ‘7 or more glasses per day’ for some beverages. Nutrient intakes are computed from the Australian food composition database AusNut 2011–2013 primarily and AusFoods (Brands) Revision 5 (Australian Government Publishing Service, Canberra).

Socio-demographic characteristics and clinical information

Sociodemographic characteristics are obtained as part of the RAW questionnaire in assessment session 1 which includes factors such as level of education, employment status, ethnicity, alcohol consumption (AUDIT-C) and smoking status.

Data management plan

All data collection procedures are conducted according to the study Standard Operating Procedure (SOP) manual. All research staff involved in a data collection procedure are appropriately trained and assessed in competence by the Principal Investigator. Intertester and intratester reliability is assessed for all individuals involved in the data collection procedure. The Principal Investigator performs quality control activities to verify study data and ensure data are complete and accurate. In addition, study-related procedures and processes, including adherence to SOPs are monitored to ensure alignment with the approved protocol, Good Clinical Practice (GCP) and institutional guidelines. The study case report forms (CRFs) are the primary data collection instruments for this study, therefore the accuracy, completeness, legibility and timeliness of the data reported is essential. All data requested on the CRF has to be recorded, missing data are explained. Error entries are corrected by drawing a single straight line through the incorrect entry so as not to obliterate the original entry and the correct data are entered above it. Changes are initialled and dated.

Information about the participants in the trial are obtained and stored in paper and electronic file format throughout and after the study. In accordance with GCP guidelines the trial files and documents are kept in a secured filing area. The storage area is locked and accessible to staff who have security access. All electronic databases are password protected. Reported study data are verifiable from the source documents. Source data includes all information, original records of clinical findings, observations or other activities in clinical trials necessary for the reconstruction and evaluation of the trial. Source data are contained in the original source documents.

Ethics and dissemination

The LIFE study protocol was approved by the University of the Sunshine Coast Human Research Ethics Committee (ethics approval number #S221718). Study findings will be disseminated through peer-reviewed journal articles, national and international conference presentations and community stakeholder engagement. Data supporting the findings of this study will be made available on reasonable request to the corresponding author, with supporting data and findings accessible within published articles and supplementary materials. There are no significant harms or risks associated with participation in the LIFE study. The procedures used are designed to have minimal risk to participants. Participants may experience some minor and transient issues with the assessments, which are described below:

DXA is a routine measurement for bone density and body composition and the associated dose with DXA (GE Lunar iDXA) was 3 µSv for the whole body. In comparison, an individual receives between approximately 4 and 5.5 µSv for daily natural background exposure, 80 µSv for a return trans-Pacific flight and 100 µSv for a chest X-ray. Therefore, although ionising radiation is used in the scan the corresponding risk from participating in this study is low. In addition, assessment and approval for the use of DXA ionising radiation from The University of the Sunshine Coast Radiation Safety Officer was obtained prior to commencement of this study.

All venous blood sampling procedures are carried out by a qualified phlebotomist to minimise risk of bruising, and all occupational health and safety procedures are followed to minimise the risk of infection associated with venous blood sampling. All blood sampling procedures are carried out in dedicated laboratory spaces.

The risk of misuse of genetic information derived from this study is very low. Breach of confidentiality may occur during data collection or biological sample collection procedures. Data and biological samples are stored in accordance with the Australian Privacy Guidelines (April 2014) and the National Health and Medical Research Council Statement on Ethical Conduct in Human Research (2007).

Completing questionnaires may be stressful for some participants depending on their cognitive status, mood, willingness, etc. Care is taken when administering questionnaires and if any stress or discomfort is observed, participants are given the option of returning to the test/questionnaire at a later time.

Clinical laboratory abnormalities are documented as adverse events if any one of the following conditions was met: (1) the laboratory abnormality is not otherwise refuted by a repeat test to confirm the abnormality; (2) the abnormality suggests a disease and/or organ toxicity; and the abnormality was of a degree that requires active management, for example, more frequent follow-up assessment, further diagnostic investigation by a medical professional etc.

Questionnaire values are documented as adverse events if any of the following conditions are met: (1) extremely high and low scores are identified and monitored, and discussed with the participant for appropriate referral to their treating medical practitioner; or (2) anxiety or stress caused by completion of study surveys is documented as an adverse event and the participant is recommended to visit their GP.

Information on all adverse events is recorded immediately in the source document and in the appropriate adverse event module of the case report form. All clearly related signs, symptoms and abnormal procedural results are recorded in the source document. The clinical course of each event is followed until resolution, stabilisation, or until it is determined that study participation was not the cause.

All serious adverse events are reported within 24 hours of occurrence, in accordance with the study protocol and GCP guidelines. Serious adverse events are defined as events resulting in an inability to carry out usual activities and are reported to the HREC through the appropriate pathways. The investigator keeps a copy of the procedure on file at the study site.

Statistical analysis plan

Data will be analysed using SPSS (V.22.0, SPSS, Chicago, Illinois, USA) and Prism (V.7.0, GraphPad, San Diego, California, USA) statistical software packages. Data will be analysed per protocol, and participants with missing data points will be excluded from formal analysis. General linear models including analysis of variance, linear regression and generalised linear models (where appropriate) will be used to address study aims. All assumptions will be tested, and statistical significance will be set at p<0.05.

Data availability

Data supporting the findings of this study will be made available on request to the corresponding author. The authors confirm that the supporting data and any subsequent findings from this study will be available within published articles and supplementary materials.

Pilot results

Recruitment for the LIFE study commenced in October 2022 with data collection and analysis expected to be completed by April 2026. Preliminary participant characteristics are summarised in table 1. Participants (n=165) are distributed across premenopausal (n=43), perimenopausal (n=55) and postmenopausal (n=67) stages. The target sample size of n=175 calculated from the power level of 0.95 has not been reached. However, a sample size calculation with power level reduced to 0.90, alpha level of 0.05, determined n=144 (48 per group) is sufficient. Although the number of participants exceeds 144 it is of note that the premenopausal group is slightly below target, which must be considered when discussing future analysis and results.

Table 1. Participant characteristics.

	All (n=165)	Pre (n=43)	Peri (n=55)	Post (n=67)
Age, mean (SD)	51.30±6.82	45.04±3.29	48.12±3.69	57.93±4.36
Ethnicity, n (%)
Australian	143 (87)	34 (79)	46 (84)	63 (94)
Aboriginal/Torres Strait Islander	3 (2)	2 (5)	1 (2)	–
New Zealander	4 (2)	2 (5)	1 (2)	1 (1)
North-West European	16 (10)	7 (16)	7 (13)	2 (3)
Southern and Eastern European	1 (1)	–	–	1 (1)
North-East Asian	2 (1)	1 (2)	1 (2)	–
Peoples of the Americas	3 (2)	2 (5)		1 (1)
Other	6 (4)	1 (2)	3 (5)	2 (3)
Marital status, n (%)
Married or partnered	132 (80)	36 (84)	44 (80)	52 (78)
Single (never married)	15 (9)	1 (2)	7 (13)	7 (10)
Separated or divorced	16 (10)	6 (14)	4 (7)	6 (9)
Widowed	2 (1)	–	–	2 (4)
Household, n (%)
My partner/spouse	125 (76)	34 (79)	39 (71)	52 (78)
My children	95 (58)	40 (93)	41 (76)	14 (21)
Parent(s)	7 (4)	1 (2)	1 (2)	5 (7)
Other	6 (4)	–	2 (4)	4 (6)
Live alone	12 (7)	–	3 (5)	9 (13)
Education level, n (%)
Bachelor’s degree or higher	94 (57)	28 (65)	39 (71)	27 (40)
Certificate/diploma	44 (27)	11 (26)	10 (18)	23 (34)
Left school after age 16	10 (6)	2 (5)	2 (4)	6 (9)
Left school at 16 years or less	8 (5)	–	1 (2)	7 (10)
Other	9 (6)	2 (5)	3 (5)	4 (6)
Employment status, n (%)
Working full-time	56 (34)	17 (40)	22 (40)	17 (25)
Working part-time	58 (35)	16 (37)	22 (40)	20 (30)
Working more than one job	17 (10)	3 (7)	8 (15)	6 (9)
Student	21 (13)	11 (26)	10 (18)	4 (6)
Homemaker	19 (12)	10 (23)	6 (11)	3 (4)
Unemployed	1 (1)	–	–	1 (1)
Retired	9 (6)	–	–	9 (13)
Volunteer	6 (4)	2 (5)	1 (2)	3 (4)
Other	22 (13)	3 (7)	4 (7)	15 (22)
Work schedule, n (%)
Regular day shifts	102 (62)	30 (70)	39 (71)	33 (49)
Regular evening shifts	1 (1)	–	1 (2)	–
Rotating night shifts	2 (1)	1 (2)	–	1 (2)
Other	20 (12)	4 (9)	6 (10)	8 (12)
Smoking status, n (%)
Person who smokes currently	2 (1)	–	1 (2)	1 (2)
No, but used to smoke	53 (32)	14 (33)	16 (29)	23 (34)
No, never smoked habitually	110 (67)	29 (67)	38 (69)	43 (64)
Alcohol intake, n (%)
Never	14 (9)	1 (2)	9 (16)	4 (6)
Monthly or less	33 (20)	13 (30)	10 (18)	10 (15)
2–4 times per month	33 (20)	10 (23)	11 (20)	12 (18)
2–3 times per week	51 (31)	12 (28)	20 (36)	19 (28)
4+ times per week	34 (21)	7 (16)	5 (9)	22 (33)

Participant characteristics detailing age, ethnicity, marital status, education level and occupation information. Participants were able to select multiple responses for employment status, household and ethnicity.

The majority of participants identify as Australian (87%), with smaller proportions identifying as European (10%), New Zealander (2%), Peoples of the Americas (2%), Aboriginal and/or Torres Strait peoples (2%) and other ethnic groups (1% each for Asian and Southern and Eastern European). Most participants are married or partnered (80%) and live with their spouse (76%), with similar proportions across groups. The majority of participants have either a bachelor’s degree or higher (57%), although there is some difference in education level between pre (65%), peri (71%) and postmenopause (40%) groups. Employment data revealed that most participants are either working full or part time (34% and 35%, respectively), with similar distributions across groups aside from the number of participants who identified as ‘Homemaker’ (pre 23%, peri 11% and post 4%). This is most likely due to the average age of participants across the groups. The majority of participants have never habitually smoked (67%) and drink alcohol between 2 and 4+ times a week (52%). There were no adverse events reported to-date in this study.

Implications for future research

The primary objective of the LIFE study is to develop an understanding of how lifestyle behaviours, such as sleep, physical activity, dietary intake and pro-inflammatory cytokines differ between pre, peri and postmenopausal women. The findings from this study will inform future research in the prevention of chronic inflammatory conditions and direct lifestyle interventions for midlife and older women at risk of obesity-related chronic disease.

The secondary objective is to identify whether differences in lifestyle behaviours and pro-inflammatory cytokines in pre, peri and postmenopausal women are associated with visceral adiposity. Investigating these relationships will support future research examining the mechanisms linking central adiposity and inflammation across the menopausal transition. A further objective is to investigate whether differences in pro-inflammatory cytokines and visceral adiposity are associated with cognitive function and well-being (depression, anxiety and stress measures) and menopausal symptoms. Understanding these relationships will assist in the development of strategies to mitigate cognitive decline and improve psychological health in midlife and older women.

Strengths and limitations

The strengths of the study include the comprehensive assessment of both behavioural and biological factors, incorporating multiple measures of key lifestyle behaviours such as sleep, physical activity, sedentary behaviour and dietary intake, including device-based assessment where applicable, alongside detailed assessment of inflammatory cytokines and body composition. This multidimensional approach enables a more integrated understanding of the relationships between lifestyle, inflammation, adiposity and cognition across the stages of menopause. Furthermore, the recruitment of participants from a regional area allows translation of findings to non-metropolitan settings.

Several limitations should be acknowledged. The cross-sectional design precludes the inference of causality and limits the ability to capture change over time. Longitudinal studies are therefore required to characterise lifestyle behaviours, inflammation and adiposity, and clarify their contribution to disease risk and cognitive function at midlife. In addition, although age will be considered a covariate, disentangling the independent effects of chronological and reproductive ageing remains challenging in midlife cohorts. Future research may benefit from incorporating narrower age ranges to minimise residual confounding.