The Role of Diet Quality in Sleep Among Women in Postmenopause (EMILE)

Participants assigned to this arm will follow a high-fiber Mediterranean diet targeting a daily dietary fiber intake of 30-35 g for 16 weeks. They will receive individualized nutritional counseling, a personalized weekly meal plan, and intensive follow-up to support adherence to the dietary recommendations.

Consists of an intensive nutritional intervention based on a high-fiber Mediterranean diet (target intake: 30-35 g/day of dietary fiber).

Participants assigned to this arm will receive low-intensity nutritional guidance on following a Mediterranean diet for 16 weeks. They will be provided with educational materials on Mediterranean dietary recommendations, without specific dietary fiber intake targets or personalized weekly meal plans.

Consists of receiving low-intensity nutritional counseling on the Mediterranean diet.

The Spanish version of the Pittsburgh Sleep Quality Index (PSQI) will be used to assess subjective sleep quality. The global score is calculated by summing seven component scores, resulting in a total score ranging from 0 to 21. Higher scores indicate poorer sleep quality.

Defined as the length of time, in min, it takes to transition from wake to sleep onset. Measured with an actigraph (ActTrust, CONDOR, Brazil)

Calculated in percentage as the ratio of total sleep time (TST) to time in bed (TIB).

Sleep efficiency (percent) = (TST/TIB)*100

Measured in minutes of WASO with an actigraph (ActTrust, CONDOR, Brazil)

Quantified using urine Melatonin-Sulfate ELISA kit

The Epworth Sleepiness Scale (ESS) will be used to assess daytime sleepiness. The questionnaire consists of eight items, each scored from 0 to 3, yielding a total score ranging from 0 to 24. Higher scores indicate greater daytime sleepiness.

Calculated as weight (kg) divided by height (in squared meters), BMI= kg/m^2

Measured as the percentage of body fat with a body composition analyzer (Inbody 120, Korea)

Measured midway between the lower rib margin and the iliac crest with the subject standing and wearing only underwear, at the end of gentle expiration with an anthropometric tape (CESCORF, Brazil)

Measured at the level of the greater trochanter, at the widest portion of the buttocks with an anthropometric tape (CESCORF, Brazil)

The composition and functionality of the gut microbiota will be assessed in collected samples through sequencing of the 16S rRNA gene fragment (V3-V4 region). Sequencing will be performed using the Illumina MiSeq 300 × 2 platform (Illumina Inc., San Diego, CA, USA), and sequence reads will be merged and processed using the MiSeq Run and MiSeq Reporter software packages.

From this data we will estimate:

• Alpha diversity: defined as the number of observed operational taxonomic units (OTUs; representing richness), through Pielou's evenness index and Shannon diversity indices.
• Beta diversity will be assessed using the unweighted UniFrac distance.
• Taxonomic assignment of phylotypes will be performed using a Bayesian classifier trained on the SILVA database, version 132, which includes full-length sequences clustered at 99% OTU similarity.

Intestinal permeability will be evaluated by measuring two widely used biomarkers, calprotectin and zonulin, in fecal samples using ELISA kits (MyBioSource, San Diego, CA, USA). Lower concentrations of calprotectin and zonulin indicate improved intestinal barrier function and suggest reduced intestinal permeability.

SCFAs will be quantified will be quantified using a headspace gas chromatography-mass spectrometry (HS-GC-MS)-based method. Higher concentrations of short-chain fatty acids, particularly butyric acid, will be indicative of greater dietary fiber intake.

Serum levels of Glucose (mg/dL) will be determined according to the procedures and protocols established by the Biochemical Analysis Laboratory of Sant Pau Hospital.

Serum levels of Total cholesterol (mg/dL) will be determined according to the procedures and protocols established by the Biochemical Analysis Laboratory of Sant Pau Hospital.

Serum levels of High-density lipoprotein cholesterol (HDL-C) (mg/dL) will be determined according to the procedures and protocols established by the Biochemical Analysis Laboratory of Sant Pau Hospital.

Serum levels of Low-density lipoprotein cholesterol (LDL-C) (mg/dL) will be determined according to the procedures and protocols established by the Biochemical Analysis Laboratory of Sant Pau Hospital.

Serum levels of Triglycerides (mg/dL) will be determined according to the procedures and protocols established by the Biochemical Analysis Laboratory of Sant Pau Hospital.

Serum levels of Glycated hemoglobin (HbA1c) (%) will be determined according to the procedures and protocols established by the Biochemical Analysis Laboratory of Sant Pau Hospital.

Diet quality will be assessed using the Mediterranean Diet Adherence Screener, which has been validated for the Spanish population. This questionnaire evaluates dietary habits such as daily consumption of fruits and vegetables, regular intake of legumes and fish, and preference for olive oil as the main culinary fat. Each item is scored as either 0 or 1, depending on whether the response is negative (0) or affirmative (1). Based on the total score, adherence to the Mediterranean diet is classified as low (0-5 points), moderate (6-9 points), or high (≥10 points).

This variable will be assessed using the Three-Factor Eating Questionnaire (TFEQ-R21C). This questionnaire consists of 21 items designed to evaluate the following dimensions of eating behavior:

• Cognitive restraint, which refers to an individual's conscious effort to control food intake in order to maintain or lose weight.
• Uncontrolled eating, which reflects the tendency to overeat in response to a perceived loss of control over eating.
• Emotional eating, which refers to the tendency to overeat when an individual is unable to cope with negative emotional situations or mood states.

Scores for each dimension are calculated as the mean of all items within that dimension, with higher scores indicating a greater expression of the corresponding eating behavior.

These outcomes will be assessed using a computerized test battery administered with E-Prime 3.0 software (Psychology Software Tools, USA). Test results will be recorded automatically by the software and exported directly to the investigator's computer. The assessment battery will last approximately 45-50 minutes and will include the following tasks:

• Rey Auditory Verbal Learning Task, which assesses verbal memory and learning.
• Corsi Blocks Task, which asesses visuospatial working memory
• Task Switching Task (TST), which assesses attention and cognitive flexibility.
• Finger Tapping Task (FTT), which assesses motor speed and dexterity.

Assessed using a visual analog or numeric rating scale integrated into the computerized battery. Participants will rate their perceived level of mental fatigue at the time of assessment.

The Hospital Anxiety and Depression Scale (HADS) consists of 14 items divided into two seven-item subscales assessing anxiety (HADS-A) and depression (HADS-D). Each subscale yields a score ranging from 0 to 21, with higher scores indicating greater severity of anxiety or depressive symptoms, respectively.

The World Health Organization-Five Well-Being Index (WHO-5) will be used to assess subjective mental well-being. The questionnaire comprises five items, each scored from 0 to 5, yielding a raw total score ranging from 0 to 25. Higher scores indicate better mental well-being.

Health-related quality of life will be assessed using the Cervantes Scale, a 31-item questionnaire specifically developed for menopausal women. The total score ranges from 0 to 155, with higher scores indicating poorer health-related quality of life.

Food noise will be assessed using the Food Noise Questionnaire (FNQ), a five-item instrument measuring the frequency and intrusiveness of food-related thoughts. Each item is scored from 0 to 4, yielding a total score ranging from 0 to 20. Higher scores indicate greater food noise

Physical activity levels will be measured in Metabolic Equivalents of Task (METs) using the short version of the International Physical Activity Questionnaire (IPAQ), validated in the Spanish population . Higher scores reflect a greater intensity of physical activity.

Additionally, physical activity levels will be objectively measured using an actigraphy device (ActTrust, Condor, Brazil), as the mean value of motor activity. Higher values reflect a greater intensity of motor activity.

Flourishing will be assessed using the eight-item Flourishing Scale. Each item is rated on a seven-point Likert scale from 1 to 7, yielding a total score ranging from 8 to 56. Higher scores indicate greater psychological flourishing.

Dietary intake will be evaluated throug a 3-day image-based dietary records. Data from dietary records will be processed by a registered dietitian using PCN Pro 1.0 software to estimate the average daily energy (kcal/day) and nutrient intake.

Adherence to the intervention will be subjectively assessed by the dietitian using a 4-point ordinal scale:

• 3 = Very good adherence: Consistent adherence to the dietary recommendations.
• 2 = Good adherence: Occasional deviations from dietary recommendations.
• 1 = Moderate adherence: Adherence during weekdays but not on weekends.
• 0 = Poor adherence: Failure to follow the dietary recommendations.

This outcome will be measured through the Invisible Family Load questionnaire to measure the:

• Managerial
• Cognitive
• Emotional load activities to address family's needs, goals, activities, responsibilities, and/or well-being. Higher scores indicate higher managerial, cognitive, and/or emotional load.

Handgrip strength will be assessed using a Jamar Hydraulic Hand Dynamometer. Three maximal grip-strength measurements will be obtained from the dominant hand following a standardized protocol, and the highest value (kg) will be used for analysis.