Plans for assessment and collection of outcomes

Assessment and data collection at baseline and follow-up will be conducted at RU1 and RU2 by trained study staff. Subjects will undergo examination between 7:30 am and 10:30 am following a 12-h fasting period. Initially, participants will receive detailed instructions about the trial’s objectives and methodologies. The standardized baseline assessment for both groups will encompass a questionnaire addressing demographic information, risk factors, comorbidities, and the NOVA Food Frequency questionnaire (NFFQ)—a validated tool for estimating UPF consumption in the Italian population [20]. Participants will complete a 3-day weighted dietary record (two weekdays and one weekend day) to enhance the accuracy of energy and nutrient intake estimation. A dietitian, utilizing specific nutritional software linked to a country-specific food-nutrient database, will retrieve these data.

Anthropometric measurements, including height, weight, and BMI, will be recorded using a stadiometer and a professional weighing scale. Furthermore, measurements of waist and hip circumferences will be taken, with subsequent calculation of the waist-to-hip ratio. Body composition will be assessed using a bioelectrical impedance analyzer. Blood pressure will be measured with a sphygmomanometer. Blood, urine, and fecal samples will be collected at baseline and follow-up visits for subsequent analyses of biochemical, inflammatory, oxidative stress markers, and microbiota-associated markers.

At the conclusion of the visit, intervention diets will be dispensed by trial personnel. All participant sessions will maintain uniformity in duration and content. Nutritionists will be instructed not to express favoritism toward either diet or disclose personal eating habits.

Plans to promote participant retention and complete follow-up

Strategies to enhance participant retention and follow-up completion incorporate behavior change methodologies, including self-monitoring, alongside continuous availability of study staff for dietary counseling. Upon study completion, participants will be requested to complete an adherence questionnaire, providing insights into their compliance with assigned diets, modifications made, and encountered difficulties. Adherence to the MD will be assessed using the MEDI-LITE adherence score [21] at baseline and during follow-up visits. Participants achieving ≥ 10 points on a scale ranging from 0 to 18 will be considered adherent [22]. For assessing compliance to UPF consumption, food diaries will be evaluated.

In cases where participants miss scheduled appointments, up to three phone calls and an email will be initiated before considering withdrawal from the study. Participants opting for premature discontinuation will not undergo further clinical and laboratory evaluations. The reasons for withdrawal will be documented for subsequent analysis during result interpretation. The entire study will be discontinued if observed results necessitate premature discontinuation.

Data management

All data will be systematically recorded in an electronic database. Participants will be identified solely by a unique study identification number to maintain pseudonymization, and no personally identifiable details will be stored in the database. To ensure data quality, various strategies will be employed, including meticulous recruitment, a structured and time-limited protocol, a run-in period, minimized participant burden, and the establishment of a trusting relationship between research units and participants. Additionally, double data entry will be implemented. Biological samples will be stored under optimal conditions following standard procedures. Blood samples will be aliquoted and preserved at − 20 °C for up to 5 years, with proper documentation of their usage or destruction. Preserved samples will be exclusively utilized for research purposes with the donor’s consent. Destruction of samples will be properly documented. The data will be made available upon request after publication.

Confidentiality

Identifiable details will be safeguarded through the allocation of unique study identification numbers, ensuring anonymity in data storage and analysis. Hard copies linking participant identification numbers to contact details will be stored in a locked file cabinet within a secure office. Access to this information will be restricted to key members of the research team. Participant files, source data, and related study documents will be retained for 5 years, the maximum duration allowed by the institution.

Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use

Blood, fecal (four or five scoops totaling 4 g), and urine samples will be collected at the beginning and end of each intervention phase.

Analysis of biochemical parameters, adipokines, and inflammatory markers (RU1, RU2, and RU3)

Blood samples will be centrifuged at 3000 rpm for 15 min to obtain serum and plasma. Samples will be aliquoted and stored at − 20 °C until analysis. Biochemical parameters (complete blood count with formula, lipid and glycemic profiles, vitamin and mineral profile) will be evaluated in all participants according to standardized routine laboratory protocols. Fasting insulin secretion capacity will be evaluated as the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) according to the related equation. Fasting plasma concentrations of ghrelin, leptin, adiponectin, resistin, visfatin, and plasminogen activator inhibitor 1 (PAI-1) will be measured using commercial enzyme-linked immunosorbent assay kits, according to the manufacturer’s instructions. Pro-(anti)-inflammatory cytokines (e.g., interleukin (IL)-1ra, IL-4, IL-10, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNFα)) will be analyzed in plasma according to manufacturers’ instructions.

Analysis of oxidative stress to macromolecules (RU1, RU3)

The 5-, 8-, 12-, and 15-F2-isoprostanes will be detected and quantified in urine samples through a high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method [23]. Protein and additional markers of lipid damage (e.g., oxidized LDL) will be investigated through analysis of protein carbonyls in serum samples by using specific enzyme-linked immunosorbent assay (ELISA) assays. The evaluation of deoxyribonucleic acid (DNA) damage will employ the Comet assay, specifically focusing on hydrogen peroxide (H2O2)-induced DNA strand breaks and endogenous oxidized DNA bases, providing insights into oxidative stress [24].

Analysis of markers of endothelial and intestinal permeability (RU1)

Endothelial permeability will be measured through the analysis in serum samples of several markers (e.g., VE-cadherin, occludin (OCLN), and claudin-5 (CLDN-5)), while intestinal permeability will be assessed by measuring zonulin, calprotectin, and tight junction proteins in serum and fecal samples. Analysis will be performed by using ELISA assays.

Analysis of fecal microbiota profiles and SCFAs (RU2)

Fecal sample collection kits, including containers, will be provided to each participant. Fecal microbiota profiles and SCFAs (acetic, butyric, isobutyric, propionic, valeric, and isovaleric acids) will be evaluated. Total microbial DNA will be extracted from the faces by repeated scouring. The V3 and V4 hypervariable regions of the 16S rRNA gene for bacteria and ITS1-4 for fungi will be sequenced on the Illumina MiSeq platform, following the Illumina protocol for preparing 16S metagenomic sequencing libraries. SCFAs will be extracted using aqueous sodium hydroxide (NaOH) containing an internal standard. After extraction, an aliquot of supernatant fecal water will be derivatized with a propanol/pyridine mixture. The organic extract will be analyzed by gas chromatography-mass spectrometry (GC–MS) using deuterated internal standards and an appropriate GC Wax column.

Analysis of fatty acid composition of red blood cell phospholipids (RU1)

From the tube containing plasma, the buffy layer of white blood cells will be removed using a pipette. Red blood cells (RBCs) will be washed twice in an equal volume of a physiologic solution (0.9% NaCl, w/v). Two aliquots (0.5 mL) of RBCs will be stored at − 80 °C until the analysis. Extraction of RBC phospholipids will be carried out in accordance with the method previously described by Simonetti et al. [25]. The FA composition of RBCs will be obtained by GC analysis following the method described by Ackman [26].

Measurement of food waste and metabolic food waste (RU1, RU3)

Food waste will be monitored by a specific food diary [27]. Subjects will be asked to fill a 3-day weighed food waste diary for each intervention arm, by registering type and weighed amount of waste. Waste will be quantified not only as a net amount of food waste (grams per week of total waste and for each food group) but also performing a nutritional and environmental assessment of food wasted [28].

The Metabolic Food Waste indicator [MFW (kg of food)] corresponds to the amount of food leading to an excess of body fat and its impact on the environment expressed as carbon [MFW (kgCO2 eq)], water [MFW(× 10 L)], and land footprint [MFW(× 10 m2)] [29]. Calculations will be applied in agreement with our previous publication [29] with few modifications, including data from new databases recently developed [28]. Briefly, the dietary intakes of volunteers will be analyzed and aggregated based on similar macronutrient composition. Then, the energy contribution of each food/food category to the daily energy intake will be calculated. Fruits and vegetables will be excluded because they are low energy dense foods not contributing to obesity. Then, anthropometric measurements relative to body fat mass will be considered. The difference between individual and average body fat mass will be multiplied for energy content of 1 kg of body fat to reach the total energy from exceeding body fat and distributed among the different foods according to their percentage contribution to total energy intake. The acquired data will allow us to calculate the carbon, water, and land footprints.

Calculation of the inflammatory index of foods (RU3)

The pro-(anti)-inflammatory effect of the tested food samples will be assessed after a simulated in vitro human digestion and, consequently, their exposure in a human culture cell-based in vitro model will be assessed, with the production of a small database of results for NII development. Aliquots of pairs of foods belonging to the same food group but of different NOVA categories (e.g., NOVA 1 versus NOVA 4) will be processed, i.e., washing, cleaning, cutting, mincing, and undergo cooking procedures, to be used for a human simulated in vitro digestion process, following the protocol by Minekus et al. [30]. In detail, time-dependent subsequent oral, gastric, and intestinal phases of digestion will be applied by mixing foods with solutions of digestive enzymes and buffer solutions for the digestion of carbohydrates, lipids, and proteins. Digested samples obtained during the time will be aliquoted and stored at − 20 °C till analysis.

Concerning the anti-(pro)-inflammatory effects of such digested foods, the digested samples will be incubated with innate immune cells (THP-1). Three concentrations of digesta will be used based on preliminary experiments, to identify a dose response effect, assessing the inflammatory response in the supernatants by ELISA assays after 24 h [31]. The inflammatory response will be measured in terms of 4 pro-inflammatory and anti-inflammatory factors’ production, such as inflammatory chemokines (IL-8), inflammatory cytokines (TNFα), and anti-inflammatory cytokines (IL-10, transforming growth factor-β (TGF-β)) [31]. The inflammatory response of the THP-1 to digested food samples will be compared to the response to a prototypical inflammatory stimulus (i.e., lipopolysaccharides (LPS)), used as reference control. To define the pro or anti-inflammatory unbiased effects due to digested samples and not to endotoxin contamination, the amount of endotoxin (e.g., LPS) in the digested samples will be evaluated by the chromogenic Limulus amoebocyte lysate assay [32, 33].