This study aims to improve how we understand and manage blood sugar responses in adults without diabetes. Even in people who appear healthy, blood sugar levels after meals can behave in different ways. These patterns may help predict future risk of diseases such as type 2 diabetes or other cardiometabolic problems.

To study this, researchers at IMDEA Nutrition have developed a computer algorithm called GLIA, which uses artificial intelligence (AI) to analyze continuous glucose monitoring (CGM) data. The goal is to classify people into different "glucotypes", meaning typical patterns of how their blood sugar behaves throughout the day. These glucotypes could help tailor dietary recommendations in the future.

Goals of the study

1. Train and validate the GLIA algorithm** in a large and diverse sample of adults.
2. Study how glucotypes relate to health indicators**, such as blood pressure, body composition, cholesterol, or lifestyle.
3. Predict how each person responds to different foods**, to support personalized nutrition advice.

Who can participate?

Adults 18-70 years old who:

• Do not*have diagnosed diabetes or serious metabolic disease.
• Agree to wear a glucose sensor for 14 days.
• Can keep stable eating habits and record diet and physical activity.

What participation involves

The study lasts 3 weeks and includes 3 visits:

Visit 1 - Screening (20 min):

• Review of eligibility criteria.
• Explanation of the study.
• Signing informed consent.
• Visit 2 - Initial assessment (45 min)
• Collection of personal and health information.
• Measurements: weight, height, waist, body composition, blood pressure.
• Placement of a FreeStyle Libre 3 CGM sensor.
• Instructions for:
• Completing two 3-day food records (one each week).
• Taking photos of all meals.
• Reporting physical activity.

Continuous monitoring (14 days)

Visit 3 - Final evaluation (45 min)

• Review of diet records.
• Repeat measurements.
• Blood and urine samples are collected for metabolic and molecular analyses.

Meal photos are analyzed using an AI-based food recognition model. The system identifies foods and estimates nutrients (macronutrients, vitamins, minerals, glycemic index, etc.). This helps researchers understand how meals relate to blood sugar patterns.

Potential benefits: Although participants may not receive direct health benefits, the study will:

• Improve understanding of how healthy people process glucose.
• Help identify early risk markers for metabolic diseases.
• Contribute to developing **personalized nutrition tools** based on individual glucose responses.

Risks: are minimal and mainly include:

• Mild skin irritation from the CGM sensor.
• Temporary discomfort from blood draw.

The Food_iSense Analytics (FiS) study is an observational, cross-sectional protocol designed to advance precision nutrition through the integration of continuous glucose monitoring (CGM), artificial intelligence (AI), and comprehensive phenotyping. The project builds upon preliminary work using data from the ENSATI and TEMPUS studies, where the research team developed GLIA, an AI-driven algorithm capable of generating individualized glucotypes-patterns of glycemic behavior that reflect the dynamic response of glucose to daily living conditions and meal intake.

Scientific Background and Rationale Although individuals without diagnosed diabetes may exhibit blood glucose values within standard reference intervals, the shape, duration, and variability of glucose excursions reflect underlying physiological regulation and may reveal early signs of metabolic dysfunction. Research has demonstrated high inter-individual variability in glycemic responses to identical meals, suggesting that dietary guidelines must move toward personalization.

The introduction of CGM devices (FreeStyle Libre 3) allows for high-resolution temporal data capturing minute-to-minute changes in interstitial glucose. However, traditional CGM metrics (mean glucose, time in range, coefficient of variation) do not sufficiently capture the full complexity of glucose dynamics.

GLIA addresses this limitation by extracting multidimensional features that quantify:

Peak morphology: slope, amplitude, recovery time, decay kinetics. Variability features: short- and long-term variability indexes, glycemic volatility, post-prandial oscillation density.

Chrononutrition-related features: differences in glycemic control across circadian windows (morning/afternoon/evening), alignment with habitual eating patterns.

Derived metrics: composite indexes generated via principal component analysis (PCA) and clustering.

Using machine learning and unsupervised clustering with bootstrapping, GLIA identifies stable glucose response phenotypes. These glucotypes are then examined in relation to health indicators, dietary patterns, and predictive models of individual glycemic responses.

Study Structure and Workflow Overview

The study consists of three in-person visits across approximately 21 days, during which participants undergo:

Initial assessment (demographics, anthropometrics, medical history, baseline health measures).

Continuous 14-day CGM period with detailed dietary monitoring using:

Two structured 3-day dietary records Automated AI-based food image recognition Mediterranean diet and ultraprocessed food questionnaires Physical activity questionnaires

Final assessment including biological sample collection (fasting blood and first-morning urine), updated anthropometry, and final quality check of all dietary records.

The final dataset incorporates more than 140 nutritional variables per food item, combined with high-resolution glucose time-series data, clinical phenotype, and multiple molecular biomarkers.

Registry-Related Quality Procedures and Data Governance Although this study is not a patient registry in the classical sense, the research team implements registry-grade data management procedures due to the scale, multidimensionality, and long-term value of the dataset. The following subsections reflect the registry-quality framework.

1. Quality Assurance Plan

   A comprehensive quality assurance (QA) plan governs all activities from recruitment to data analysis. Key components include:

   Standardized training of all personnel (dietitians, research nurses, data managers).

   Calibration schedules for anthropometric devices (stadiometer, scale, bioimpedance instruments) and for blood pressure monitors.

   Daily consistency checks of CGM data uploads. Protocol deviation logs documenting missing measurements, device issues, and participant non-compliance.

   Internal monthly audits performed by the IMDEA Quality Office to verify protocol adherence.

   Independent external audit capability is maintained, though audits are not routinely scheduled unless required by funders or ethics committees.

2. Data Validation and Automated Data Checks

   Incoming data are processed through a multi-stage validation pipeline:

   1. Range and plausibility checks Automatic filtering flags values outside expected biological ranges (e.g., impossible BMI, extreme macronutrient percentages, duplicate CGM timestamps).

   2. Internal consistency checks

      Cross-field validation identifies inconsistencies, such as:

      caloric intake mismatching macronutrient totals, dietary patterns incompatible with photographed meals, anthropometric values inconsistent across visits.

   3. Technical consistency

   CGM streams are checked for:

   signal dropouts longer than 15 minutes, abrupt shifts indicating sensor displacement, unrealistic glucose kinetics (rise/decay rates).

   Problematic sections are annotated but not deleted, preserving data integrity for sensitivity analyses.

3. Source Data Verification (SDV)

   To ensure data accuracy and representativeness:

   Dietary records are cross-validated against food photographs and questionnaire responses.

   Medical history and medication data are verified against documents participants bring to Visit 2 (e.g., lab reports ≤ 6 months old).

   Blood pressure and anthropometry undergo dual measurement with two assessors performing random checks on 10% of sessions.

   CGM data are compared with participant logs describing sensor issues, physical activity peaks, and atypical meals.

   All verification steps follow Good Clinical Practice (GCP) documentation practices.

4. Data Dictionary and Variable Coding

The study uses an extensive data dictionary organized into modules:

Sociodemographic module

Definitions, coding schemes (e.g., ISCED for education), universe and skip patterns.

Clinical phenotype module

Standard coding for medical conditions using ICD-10 when applicable. Definitions of metabolic syndrome markers and derived variables.

Anthropometry and body composition

Measurement rules, rounding conventions, and device-specific calibrations.

CGM module

Time-series format, sampling interval, sensor metadata, derived features, preprocessing rules.

Dietary variables

Food items mapped to validated food composition databases for Spain. Nutrient coding includes standardized units and upper/lower expected ranges.

Molecular biomarkers

Units, assay platforms, storage conditions, and laboratory reference values.

The dictionary includes more than 500 entries and is version-controlled.

SOPs govern all operational and analytic tasks, including:

Recruitment and informed consent CGM application, monitoring, and removal Anthropometric and BIA measurement Blood/urine sample collection, processing, aliquoting, and storage Data entry and double-entry verification for paper forms Food image capture guidelines and data upload protocol Use of the AI food-recognition system and manual override procedures Documentation of adverse events related to CGM or blood collection Dataset freeze procedures and change control for analysis scripts Secure storage, encryption, and pseudonymization workflows

SOPs are reviewed periodically and updated as needed.

6. Sample Size Assessment

Using synthetic data generated during algorithm development, investigators determined that approximately 392 complete datasets provide adequate clustering stability:

Maximization of the Silhouette score (~0.67 at n=392). Near-minimal Davies-Bouldin index at ~392-588 samples. Robust PCA component stability at ≥350 participants.

Accounting for a 20% potential data-loss rate, 471 participants are targeted for recruitment.

7. Missing Data Plan Missingness may arise from incomplete dietary records, sensor failures, lost images, or participant withdrawal.

The plan includes:

Classification of missingness as MCAR, MAR, or MNAR. Short-gap interpolation for CGM data gaps ≤ 20 minutes using cubic spline or Kalman smoothing.

Exclusion of unusable segments (e.g., sensor warm-up periods). Multiple imputation (MI) for covariates such as weight or blood pressure if only one visit yields valid measures.

Complete-case sensitivity analyses to confirm robustness of imputed models.

Retention strategies minimize missing data, but high technical data density ensures analytic viability even with partial loss.

8. Statistical Analysis Plan (SAP) The SAP covers both algorithm validation and hypothesis-generating analyses. 8.1 Preprocessing

CGM normalization to individual baselines. PCA for dimensionality reduction. Unsupervised clustering (k-means and hierarchical) with bootstrapping. Feature engineering for peak characteristics and chrononutrition variables.

8.2 Glucotype generation and validation

Internal validation with Silhouette, Calinski-Harabasz, and Davies-Bouldin metrics.

Stability assessment through subsampling and perturbation tests.

8.3 Association analyses

ANOVA/Kruskal-Wallis for between-glucotype comparisons. Logistic and linear regression adjusting for key confounders. MANOVA for overall cardiometabolic profiles.

8.4 Dietary modelling

Multiple Correspondence Analysis (MCA) for dietary pattern extraction. Regression models linking nutrient intake to glucotype. Machine learning models (Random Forest, SVM, XGBoost) to predict meal-level glucose responses.

8.5 Predictive simulations Agent-based modelling is used to simulate hypothetical diet changes and predict personalized glucose responses under various nutritional scenarios.

8.6 Software and reproducibility Analyses are performed in Python and R, with scripts managed through version control, containerization (Docker), and reproducibility logs.

Conclusion The FiS protocol integrates advanced CGM analytics, AI-driven phenotyping, and detailed dietary and biochemical profiling to map the diversity of glucose regulation in adults without diabetes. The study applies rigorous registry-level quality procedures, including automated data validation, structured data dictionaries, SOPs, and a comprehensive statistical analysis plan. The resulting dataset and validated algorithm will support future work in personalized nutrition, early risk detection, and tailored dietary interventions.

Inclusion Criteria:

• Adults aged 18 to 70 years.
• Willing and able to undergo 14 days of continuous glucose monitoring (CGM) using a wearable sensor.
• Able to maintain stable dietary habits during the monitoring period.
• Able and willing to complete dietary records, including two structured 3-day food logs.
• Able and willing to photograph all meals during the 14-day monitoring period following instructions provided.
• Able to keep a record of physical activity as instructed.
• No previous diagnosis of diabetes or other serious metabolic disorders.
• Sufficient commitment and availability to attend all study visits (screening, baseline evaluation, final evaluation).
• Capable of providing written informed consent.

Exclusion Criteria:

• Diagnosed diabetes mellitus or other serious metabolic disorders.
• History of severe gastrointestinal, cardiovascular, or other medical conditions that may interfere with stable diet or physical activity during the study.
• Pregnant or breastfeeding women.
• Inability or unwillingness to comply with continuous glucose monitoring (CGM) procedures for 14 days.
• Participants with skin conditions or allergies that prevent safe use of a CGM sensor.
• Current participation in another clinical trial that could affect study results.
• Use of medications that significantly alter glucose metabolism or interfere with CGM accuracy.
• Inability to attend all scheduled study visits or complete required records (diet logs, photos, questionnaires).
• Any condition judged by the investigators to make the participant unsuitable for the study or unable to provide informed consent.

The study population consists of community-dwelling adults aged 18-70 years, recruited from the general population through public advertisements, university campus postings, pharmacies, and primary care centers. Participants represent a broad, non-clinical community sample without diagnosed diabetes or severe metabolic disease. Recruitment is open to individuals living independently and able to maintain their usual daily routines. The population reflects a heterogeneous mix of sociodemographic backgrounds to ensure variability in dietary habits, lifestyle patterns, and glucose-response profiles.

De-identified individual participant data will be made available to qualified researchers upon reasonable request. All shared datasets will undergo double codification, meaning two independent pseudonymization layers are applied before external release: one ID replacing personal identifiers within IMDEA Nutrition, and a second external-use ID generated solely for data sharing. No key linking either code to participant identities will be shared. Data will be accessible only for ethically approved scientific purposes and after signing a data-sharing agreement outlining permitted use, data-security requirements, and obligations to prevent re-identification. Access will be provided through secure, controlled-transfer procedures.

Individual participant data (IPD) and accompanying documentation will become available not before 12 months after completion of the final data analysis, anticipated to begin once all primary and secondary outcomes are fully evaluated. Data will remain accessible to qualified researchers for a minimum of 5 years following the initial release. After this period, continued availability will depend on dataset relevance, ethical approvals, and storage capacity. Access will be granted only through controlled procedures and under a signed data-sharing agreement ensuring secure use and strict protection against re-identification.