Vitis Intervention Trial in Substrate Metabolism

Recruitment Participants are recruited from the UAB/Birmingham catchment area (the University of Alabama at Birmingham, the city of Birmingham, and Jefferson and Shelby Counties) through research registries, clinic flyers, web-based advertisement, the Department of Nutrition Sciences website, ResearchMatch, and word of mouth. Interested individuals contact study staff for an initial study overview and eligibility screener.

Prescreening Interested individuals will contact study staff and receive an overview of the study and then provide verbal consent to answer questions for high level eligibility. This will include questions related to demographics, anthropometrics, current use of nicotine and caffeine, claustrophobia, dietary adherence, use of smart phone/tablet, prescription drugs, chronic diseases and history of recent weight changes. If the interested individual remains eligible and willing to participate, an in-person screening visit will be scheduled.

Screening Screening visits are scheduled in the morning (approximately 7 am -11 am), and participants must arrive fasted. After informed consent is provided and obtained, participants will then be asked to change into a gown for procedures of height, weight, REE by metabolic cart (Parvo TrueOne 2400), DXA (Lunar GE), blood draw to screen for WBC count, and paperwork. Participants will then be provided with a weight scale and an orientation to the Healthie app. They will receive a study-linked email address to log into Healthie, no participant data will be provided to Healthie. Once they have reviewed operations of their new scale and Healthie, they will then await WBC count confirmation and once the study team has that in place, weight stability data collection will start with a calorimeter scheduled two weeks out.

Randomization and Blinding Following confirmed weight stability, participants are randomized to a grape-first or placebo-first sequence (AB or BA). Randomization and dose preparation are performed by the metabolic kitchen, which maintains allocation concealment; participants, investigators, and all other study staff (including the interventionist) remain blinded throughout. Each dose consists of 72 g of freeze-dried whole California table grape powder or a matched placebo powder formulated to replicate the sugar, fiber, organic acid, and sensory characteristics of the grape powder without polyphenolic compounds. The grape powder is a whole-food dietary supplement; no investigational new drug designation or clinical-trial phase applies.

Study Interventions/Procedures Study Design Single-site, double-blind, placebo-controlled, crossover pilot study. Twenty adults (10 lean, 10 with overweight/obesity) complete two identical 40-hour stays (arrival evening plus one full calorimeter day) in the UAB Webb whole-room indirect calorimeter, separated by a washout period. Each participant receives both freeze-dried whole California table grape powder and a matched placebo powder in random order.

Safety During each session, participants are monitored by study staff for aberrant data (e.g., fidgeting). Hourly values of calorimeter temperature, pressure, and target metabolic rate and respiratory quotient are recorded. Safety procedures within the calorimeter are reviewed with each participant prior to the start of each calorimeter day. For medical emergencies, the laboratory acts in accordance with University policy.

Safety Procedures All calorimeter safety procedures are reviewed with each participant prior to the start of every calorimeter day. The calorimeter door can be opened from the inside at any time, and staff maintain continuous communication and monitoring through intercom, call button, and video surveillance. As a 911-response facility, trained personnel are always present during calorimeter occupancy. An AED and first-aid kit are available on site. In the event of a medical emergency, participants are promptly removed from the calorimeter, 911 is activated, and care proceeds in accordance with University policy. Environmental safety is ensured through continuous oxygen and carbon dioxide monitoring with alarm systems, and the calorimeter ventilation can be rapidly purged if needed. All safety systems are tested before each study day. Participants may request to exit the calorimeter at any time for safety or comfort. All adverse events are documented and reported per IRB policy.

Safety Training and Oversight All study personnel are trained in emergency procedures, calorimeter operations, and participant monitoring. Staff complete a pre-use safety checklist before each study day to confirm function of alarms, communication systems, ventilation, and egress mechanisms. The Principal Investigator is responsible for oversight of safety protocols, review of any adverse events, and ensuring compliance with University and IRB requirements.

PROCEDURES:

Pre- Screening: For the pre-screening, interested individuals will contact study staff and receive an overview of the study and then provide verbal consent to answer questions for high-level eligibility. This will include questions related to demographics, anthropometrics, current use of nicotine and caffeine, claustrophobia, dietary adherence, use of smart phone/tablet, prescription drugs, chronic diseases and history of recent weight changes. If the interested individual remains eligible and willing to participate, an in-person screening visit will be scheduled.

Screening Visit: At the screening visit, study staff provide a detailed overview and obtain verbal consent to collect eligibility information, including medical history, a dietary polyphenol intake assessment, and standardized health questionnaires. Eligible participants attend an in-person screening visit at the UAB Webb Building to review and sign the informed consent, after which medical history and current medications are reviewed, height and weight measured, and a smart scale issued with instructions for documenting food, sleep, and physical activity.

In Person Procedures:

Informed Consent: Prior to enrollment, informed consent is obtained in accordance with the Declaration of Helsinki and federal regulations (45 CFR 46), using the IRB-approved consent document for this study. A trained study investigator reviews all procedures, risks, benefits, alternatives, and participant rights in a private setting, confirms comprehension, and documents written consent by participant signature and date; a copy is provided to the participant.

Medical History Review and Screening: Comprehensive medical history is obtained at screening to characterize participant health status and identify exclusionary conditions.

Anthropometrics Body weight is measured with participant in gown/disposable exam shorts in triplicate after voiding, using a calibrated digital scale (precision ±0.05 kg). The weight of the gown is also measured and substrate from the total weight. If two measures vary by more than 0.2 kg, then a third measure is taken and all three are recorded. Height is measured once using a wall-mounted stadiometer. Waist circumference will be measured in triplicate using a tensiometer to the nearest tenth of a centimeter.

Body Composition Body composition (fat mass, fat-free mass, bone mineral content) is assessed via dual-energy X-ray absorptiometry (DXA). These measurements permit normalization of energy expenditure to metabolically active tissue mass and enhance reproducibility by ensuring comparable baseline conditions across repeat visits.

Energy Balance Prescription Energy and calorie targets for run-in periods are obtained using standardized, validated equations and adjusted as warranted using energy-expenditure and body-composition data.

We will use the DXA and REE metabolic cart data for the NIDDK BodyWeight Planner (Advanced and Expert Modes) to guide weight stability targets during the two run in periods.

Weight stabilization is intended to minimize variability in REE and substrate oxidation measures arising from acute changes in energy balance, thereby improving interpretation of calorimeter-derived outcomes.

Questionnaires The following standardized instruments are administered at screening to characterize phenotype as well as identify covariates and exclusionary conditions during baseline and testing.

Instrument Construct / Rationale Items/Time Cut-points Action / Exclusion Flags Time Points PROMIS Global Health (10) Overall health status; anchor for between-visit variability 10 items / 2-3 min T-score < 40 Flag for covariate; consider deferring visit if acutely ill Screening PSQI Sleep quality (past month); impacts EE/RQ and adherence 19 items / 5-7 min Global score > 5 Flag; adjust scheduling or analyze as covariate Screening PSS-10 Perceived stress; perturbs appetite/sleep/EE 10 items / 2-3 min Score ≥ 20 (moderate-high) Flag; consider deferring if acute stressor; covariate Screening IPAQ-Short Habitual physical activity; context for free-living PA vs calorimeter 7 items / 3-5 min <600 MET·min/wk = Low; ≥3000 = High Use category as covariate; verify extremes Screening Raid-FN (Ro/Allison/Indiana/Dhurandhar Food Noise Inventory, short form) "Food noise" - preoccupation, persistence, and dysphoria of food-related thoughts; context for interpreting postprandial craving/intake response 23 items / 2-3 min No established clinical cut-point; scored continuously Flag; reinforce standardized meal instructions All FCQ-Trait (Food Cravings Questionnaire-Trait) Trait-level frequency/intensity of food cravings; covariate for reward-driven eating and meal compliance 15 items / 3-5 min Score ≥ 50 → clinically relevant trait craving (validated food-addiction screening threshold) Flag; reinforce standardized meal instructions All AUDIT-C Alcohol use; affects sleep/metabolism 3 items / <2 min ≥3 (women) / ≥4 (men) Counsel reduction pre-visit; consider exclusion if heavy use Screening Caffeine Checklist Caffeine dose & timing; shifts EE/RQ & sleep 5-6 items / <2 min >400 mg/day or intake after 12:00 Instruct taper/timing; reschedule if noncompliant Screening ESS (Epworth Sleepiness Scale) Daytime sleepiness; correlate with nocturnal EE 8 items / <2 min Score ≥ 10 Flag; consider OSA workup; covariate Screening Menstrual Cycle History (brief) Cycle phase/regularity; hormones/HRT; key covariate for EE/RQ 6-10 items / 2-3 min Irregular cycles; CHC/HRT use Record phase; schedule consistently; covariate Screening Gastrointestinal Severity Rating Scale Lactose/FODMAP/spice triggers; avoid protocol-related GI confounds 8-12 items / 2-3 min Any major intolerance reported Adjust standardized meals; document All PHQ-9 Depressive symptoms; context for sleep/appetite changes 9 (or 8) items / 2-3 min Score ≥ 10; item 9 > 0 Provide resources; may defer if unstable Screening GAD-7 Anxiety symptoms; stress-related metabolic variance 7 items / 2-3 min Score ≥ 10 Provide resources; covariate Screening Berlin Obstructive sleep apnea 10 items / 5-10 min 2+ positive scores out of 3 categories Flag; consider OSA workup; covariate Screening

Bluetooth Scale and Healthie Provision and Orientation:

At the in-person screening visit, following completion of informed consent, study staff issue each participant a study-owned Bluetooth-enabled digital scale (precision ±0.1 kg) that has been verified against a laboratory reference standard prior to distribution. Staff pair the device on the participant's Healthie account on-site and confirm a successful transmission before the participant leaves the visit. Participants are oriented to the daily self-weighing procedure. Weight is taken under standardized conditions, immediately after waking, post-voiding, before food or fluid intake, wearing minimal clothing (same thing each time), throughout the 14-day run-in periods. Weight stability is defined as (1) total change < 1.0 kg across 14 days; (2) day-to-day change ≤ 100 g during days 11-14; and (3) slope of log-transformed weights < ±0.5 kg/week. Participants who do not meet all predefined weight-stability criteria after 14 days may complete an additional two-week stabilization period with enhanced dietary support. Participants unable to achieve stability within four weeks will be withdraw before calorimeter admission. For the 72 hours before each calorimeter visit, participants follow a polyphenol-light diet (avoiding berries, grapes, tea, cocoa, red wine, juices, and high-polyphenol spices) to minimize background polyphenol exposure.

Healthie is a secure, HIPAA-compliant electronic health record (EHR) and telehealth platform designed for use by healthcare and wellness providers to support patient care, data collection, and communication. The platform integrates multiple functions, including appointment scheduling, secure messaging, clinical documentation, and patient engagement tools, within a single system. Participants will access Healthie through a mobile application or web-based portal to record study-related information, including daily body weight, dietary intake, physical activity, and sleep logs. The platform also allows participants to complete questionnaires and communicate with study staff as needed. Data entered by participants are stored within a secure, encrypted environment and are accessible only to authorized study personnel. Healthie supports real-time data sharing between participants and study staff and will integrate with the study provided blue tooth scale to capture health-related metrics. The platform complies with HIPAA privacy and security standards, including data encryption and secure data transmission, to ensure the confidentiality and protection of participant information.

Habitual Dietary Intake (ASA24) Habitual dietary intake is assessed using the Automated Self-Administered 24-Hour Dietary Recall (ASA24®, National Cancer Institute). This validated web-based platform guides participants through a multiple-pass 24-h recall using standardized food probes, portion-size images, and linked nutrient databases. Participants complete recalls on non-consecutive days, including at least one weekend day. Built-in consistency checks minimize under- and over-reporting, and automated prompts capture frequently forgotten items. Data output includes total energy intake, macronutrient distribution, micronutrient content, and timing of eating occasions, enabling harmonization with calorimetry-derived measures.

Sleep and Physical Activity Diary During run-in and washout periods, participants complete a standardized daily physical activity and sleep diary documenting exercise bouts, occupational activity, sedentary behavior, and sleep-wake timing, using structured time blocks with categorical activity codes, intensity ratings, and duration. Entries are cross-validated against accelerometry and calorimeter motion sensors when available. Diaries are collected electronically through REDCap, with submissions time-stamped to enhance compliance monitoring; completeness is reviewed weekly by study staff.

Accelerometry Sleep and activity will be assessed throughout each run-in and study period using wrist actigraphy (ActiGraph GT3X+, Pensacola, FL). Raw data from the device (acceleration and ambient light) will be scored using validated algorithms in the ActiLife software for estimation of sleep and different levels of physical activity as well as inactivity. ActiLife is a secure, HIPAA compliant platform; the ActiGraph devices do not have Bluetooth enabled or geolocation capabilities. Information from daily sleep diaries will be used to enhance the accuracy of bed and waketime estimates derived from scoring algorithms applied in ActiLife. Neither raw nor scored data will be stored on ActiLife.

Home Weight Scale Participants are provided a study-issued smart scale (precision ±0.1 kg) verified against a laboratory reference standard prior to distribution. Participants weigh once daily under standardized conditions (after waking, post-void, prior to food or fluid intake, minimal clothing, same time each day). Weights transmit automatically to a secure study database (Healthie platform) for monitoring by the study dietitian. Timestamped uploads monitor compliance, and outliers (±3 SD from rolling average) are flagged for verification. These data ensure weight stability prior to calorimeter entry over the 14-day run-in. In cases of device malfunction, participants notify study staff immediately and a replacement scale is provided; all deviations and imputed values are documented.

In Person Procedures:

Standardized Meals

• Standardized run-in and calorimeter day 0 meals: 30% fat / 55% carbohydrate / 15% protein.
• Mini-meals meals: 30% fat / 55% carbohydrate / 15% protein.
• High-fat test meal: Approximately 12 kcal/kg fat-free mass (maximum 1000 kcal), containing ~ 65% fat / 25% carbohydrate / 10% protein.
• Grape or placebo powder (72 g) administered 30 minutes before the high-fat test meal.

All meals are prepared to fixed energy and macronutrient specifications by the UAB Metabolic Kitchen and consumed within a standardized ingestion window under observation. No outside food is allowed. Water intake is ad libitum but recorded; caffeine and alcohol are prohibited during calorimeter periods. For the calorimeter stays, energy balance will be determined via the Lam equation; calorie prescription will allow for additional calories to be added in order to maintain energy balance within 100 calories.

Participants will be fed the energy balance prescription via the Metabolic Kitchen provided meals described above; titration will happen at the 7 hour mark to allow for the high caloric content of the MetFlex meal.

Whole-Room Indirect Calorimetry Whole-room indirect calorimetry enables continuous, high-resolution quantification of human energy expenditure and substrate oxidation within a controlled metabolic environment. The system operates as an open-circuit indirect calorimeter in which ambient air is drawn into the calorimeter at a precisely regulated flow rate, typically controlled by mass flow controllers with sub-percent error tolerance. Exhaust air is continuously sampled and analyzed for fractional concentrations of O₂ and CO₂ using paramagnetic and nondispersive infrared analyzers, with analyzer drift corrected by periodic calibration against precision gas mixtures traceable to NIST standards. Dynamic energy expenditure is calculated from VO₂ and VCO₂ using the Weir equation, corrected for calorimeter volume, barometric pressure, and water vapor dilution. Temporal resolution of VO₂ and VCO₂ is typically 1-2 min, allowing detection of rapid metabolic transitions such as the thermic effect of food. Calorimeter leak integrity is verified prior to each study using infusion tests, ensuring stoichiometric recovery of CO₂ and O₂. Derived parameters include resting energy expenditure, 24-hour energy expenditure, thermic effect of food, and the respiratory exchange ratio (RER) time series.

Prior to calorimeter stays, participants are screened for alcohol/substance use. They may not bring in alcohol, caffeine, gum, mints, or any outside contraband that may alter metabolism. Prior to calorimeter entry, study staff inspect participants' belongings for contraband. Electronic devices may be used during daytime hours but must be silenced during sleep measures.

Participants will follow a standardized posture and activity script throughout chamber testing. Physical activity intensity will be limited to low-intensity movement, including walking within the chamber and stretching, to minimize variability in metabolic outcomes.

Room Calorimeter Physical Activity Monitoring In addition to the accelerometer, spontaneous and structured activity are quantified using a multimodal approach. Infrared motion sensors embedded in the calorimeter provide positional and movement counts, while tri-axial accelerometry worn by participants records activity intensity and duration. Activity counts are time-synchronized with VO₂/VCO₂ to attribute energy expenditure to locomotor versus non-locomotor components.

Room Calorimeter Sleep Monitoring In addition to the accelerometer, sleep is monitored continuously during calorimeter stays (approximately 22:00-06:00). Participants may not nap during the day and are given clear lights-out and lights-on instructions via intercom. Participants are asked to silence cellular devices (e.g., smartwatches, TV off) and close privacy curtains to mitigate sleep disruption. Continuous overnight VO₂ and VCO₂ are used to derive sleep EE and RER.

Biospecimens Two venous draws are obtained per 40-hour stay - fasting (Day 1, ~07:55) and postprandial (Day 1, ~21:30, ~3 h after the high-fat meal) - at 36 mL each (72 mL per stay; 144 mL per participant across both stays), drawn from an antecubital vein by a trained phlebotomist or nurse. Each draw consists of: four 4 mL CPT tubes (16 mL) for Stream A PBMC culture; and four 4 mL tubes (16 mL) for Stream B mitochondrial respirometry. PBMCs are isolated by standard density-gradient centrifugation and processed for high-resolution mitochondrial respirometry (Oroboros O2k) to assess fatty-acid-supported respiration and spare respiratory capacity. Plasma is retained for acylcarnitine profiling. Samples are labeled with a unique study code and stored at -80 °C in the restricted-access Metabolism Core Lab (Webb 337). Remaining biospecimens are biobanked for future hypothesis-driven analyses contingent on additional funding.

Postprandial blood draw timing rationale: The postprandial draw is anchored to the high-fat test meal (~3 h post-meal), not to grape/placebo powder administration, because the study's primary interest is whether grape polyphenols alter the metabolic flexibility (MetFlex) response to the meal itself, not polyphenol pharmacokinetics per se. A meal-anchored ~3-hour timepoint is consistent with published postprandial monocyte activation and lipemia studies, which commonly sample in the 3-4 hour post-meal window to capture peak circulating lipid exposure and monocyte/PBMC activation. Continuous whole-room calorimetry data separately capture the full RER trajectory (meal-stimulated peak, overnight sleep nadir) per the validated MetFlex approach (McDougal et al., 2025), so the single postprandial blood draw timepoint is not relied upon to capture the RER-based MetFlex outcome itself.

Method validation note: The PBMC isolation and high-resolution respirometry (Oroboros O2k) workflow is undergoing internal method validation (comparing CPT-tube versus EDTA/density-gradient separation for cell yield, viability, and compatibility with O2k chamber volume) prior to full-scale sample processing. Validated, standard-operating-procedure-documented methods will be used for all study specimens once finalized.

Urine Twenty-four-hour urine collections are obtained from a urine collection device placed in the in-calorimeter restroom. Privacy curtains are closed at every void. Participants void into pre-weighed, acid-washed, opaque collection containers; containers are pre-treated with boric acid (10 g/L) to stabilize pH and maintained at 4 °C throughout collection. Total volume is recorded gravimetrically, and completeness is verified by creatinine excretion normalized to body mass. Nitrogen excretion is quantified by elemental combustion methods, enabling calculation of protein oxidation rates when combined with indirect calorimetry data and improving the accuracy of substrate oxidation partitioning (non-protein RER adjustment).

Calorimeter Day-by-Day Procedures (40-hour stay) Arrival (Day 0, evening): Participants arrive at the Webb Calorimetry Facility the evening before the full calorimeter day. Diaries and weights are reviewed for completeness. A standardized evening meal is provided, and participants sleep in the calorimeter overnight so that sleeping RER and energy expenditure can be measured.

Day 1 (full calorimeter day): Following a wakeup call: height/weight assessment; DXA scan (fat mass and fat-free mass, ~10-15 min); resting energy expenditure via metabolic cart. The participant then re-enters the calorimeter, where 24-hour urine collection and accelerometry begin, a fasting blood draw (~07:55) is obtained, a standardized breakfast is provided, the grape or placebo powder (72 g) is administered 30 minutes before the high-fat test meal, and a postprandial blood draw (~21:30) is obtained ~3 hours after the meal. The RAID-FN and General Food Cravings Questionnaire-Trait are completed. Lights and screens are turned off at approximately 10 PM; sleeping RER and energy expenditure are measured overnight. The intervention product will be provided in pre-weighed packets and reconstituted with water immediately before administration. Participants will consume the entire dose before the high-fat test meal.

Day 2: Following a 6:15 AM wake-up call, participants remain still for a resting energy expenditure measurement, after which the 24-hour urine collection ends and they exit the calorimeter.

Calorimeter Activity Timing Grid Times are approximate and depend on the start time of laboratory procedures; minor variations are acceptable.

Table 2:

Day Time Activity Biospecimen / Notes 0 1730 Participant enters calorimeter (VO₂/VCO₂ sampled at 1-min intervals; continuous activity monitoring) - 0 1800 Measure starts; standardized evening meal (consumed within 30 min) - 0 2200 Prepare for bed - 0 2230 Lights off; no screens (overnight sleep RER) -

1 0630 Wake; exit calorimeter for DXA and cart REE -

1 0755 Re-enter calorimeter Fasting blood draw (~40 mL); start 24-h urine

1 0800 Measure starts; standardized breakfast (within 30 min) -

1 1200 Standardized lunch -

1 1800 Grape or placebo powder (72 g) -

1 1830 High-fat test meal (within 30 min) -

1 2130 Postprandial sampling (~3 h after meal) Postprandial blood draw (~40 mL)

• 2230 Lights off; no screens (overnight sleep RER) -
• 0615 Wake; void; sip water -

2 0630-0700 Resting energy expenditure measure - 2 0705 Exit calorimeter End 24-h urine collection Biospecimens Peripheral blood mononuclear cells (PBMCs) will be isolated from 32 mL of venous whole blood collected at the specified time points during the chamber protocol (See Table 2). Baseline and post ingestive PBMCs will be isolated using standard density-gradient centrifugation methods. Isolated cells will be cryopreserved in multiple sub-aliquots and stored in liquid nitrogen to support downstream mitochondrial respiration assays and exploratory molecular analyses.

Twenty-four-hour urine collections will be obtained during chamber stays using a collection device located within the in-chamber restroom. Participant privacy will be maintained using privacy curtains during all voids. Total urine volume will be recorded gravimetrically, and completeness of collection will be verified using creatinine excretion normalized to body mass.

Urinary nitrogen excretion will be quantified using chemiluminescence or elemental combustion-based methods (e.g., Kjeldahl), enabling estimation of protein oxidation rates when integrated with indirect calorimetry data. Incorporation of urinary nitrogen with VO₂ and VCO₂ measurements will allow adjustment for non-protein respiratory exchange ratio (RQ), thereby improving the accuracy of whole-body substrate oxidation partitioning. This biospecimen strategy enables mechanistic assessment of mitochondrial bioenergetics that can be directly aligned with whole-body metabolic outcomes, while preserving sample integrity for future analyses. Primary cellular outcomes will include fatty-acid-supported mitochondrial respiration and spare respiratory capacity measured by high-resolution respirometry in cryopreserved PBMCs. These phenotypes were selected for their robustness to cryopreservation, biological interpretability, and relevance to lipid oxidation during a high-fat challenge. Exploratory respirometric parameters will be analyzed to generate mechanistic hypotheses and inform future study design. PBMC mitochondrial phenotypes will be evaluated as secondary mechanistic endpoints to contextualize changes in sleep and postprandial respiratory exchange ratio. Remaining biospecimens will be biobanked for future hypothesis-driven analyses contingent on additional funding.

Washout and Crossover After the first calorimeter stay, participants resume a eucaloric diet, avoid polyphenol-containing supplements, and maintain weight stability (±1.0 kg) during washout. The 14-day weight-stabilization run-in, polyphenol-light diet, and calorimeter procedures are then repeated for the second visit with the alternate powder. After the first calorimeter stay, participants resume a eucaloric diet, avoid polyphenol-containing supplements, and maintain weight stability (±1.0 kg) during washout. During the 72 hours preceding the crossover visit, participants repeat the polyphenol-light diet. The 14-day weight stabilization run-in and all calorimeter procedures are repeated before the second chamber stay with the alternate intervention.

Sampling Methods Convenience sampling is used to recruit eligible lean adults and adults with overweight/obesity meeting inclusion criteria from the UAB/Birmingham catchment area.

Sample Size Calculation The sample size is based on detecting a clinically meaningful absolute difference in postprandial RER (ΔRER = 0.02) between grape and placebo conditions in a 2-period crossover. Assuming a within-person SD of approximately 0.03, α = 0.05, and 80% power, 18 completers are required; the study enrolls 20 to allow for attrition.

Inclusion criteria

• Men and women aged 25-60 years
• BMI 18-24.9 kg/m² (lean phenotype, n=10) or BMI 25-40 kg/m² (overweight/obesity phenotype, n=10)
• Low habitual grape consumption
• Weight stable (no change >5% in prior 6 months) and not actively attempting weight change
• White blood cell count within normal limits

Exclusion criteria

• Current smoking; drug or alcohol abuse
• Claustrophobia or psychiatric conditions that would make a 40-hour confined stay especially distressing or harmful
• Weight instability, recent/active weight-loss attempts, or structured exercise initiation during the study
• Hormone replacement therapy or medications known to affect metabolism
• Pregnant or breastfeeding
• Current or prior bulimia nervosa, binge eating disorder, or anorexia nervosa
• Grape allergy, or food allergies/intolerances precluding use of the metabolic kitchen
• Any condition that, in the PI's opinion, would make participation unsafe or could interfere with conduct/interpretation of the study