2.4.1. Anthropometric Variables

Anthropometric parameters at baseline and after interventions included the body mass index (BMI, as the body weight in kg/m2), weight loss (WL, as ([Initial BMI]—[Post-intervention BMI])), percent of total weight loss (%TWL, as ([Initial Weight]—[Post-intervention Weight])/([Initial Weight])) × 100, and the percentage excess BMI loss (%EBMIL, as (ΔBMI/[Initial BMI–25]) × 100). We evaluated systolic and diastolic blood pressures by using automatic tensiometer device (Omron M2-HEM-7121-E, Kyoto, Japan). Blood samples were centrifuged for 20 min at 2.500 *g* and the obtained plasma was tested for glucose and lipid determinations. The fasting glucose and glycated hemoglobin (A1C), and the lipid profile (triglycerides, total cholesterol, LDL-cholesterol, and HDL-cholesterol) were quantified by standard methods (ADVIA 2400 Chemistry System, Siemens, Germany) in the Analytical department of Hospital Fundación Jiménez Díaz. All variables were measured before and after interventions.

#### 2.4.2. Analysis of Microbiota

Fecal samples were obtained in OMNIgene-GUT tubes (Abyntek, Spain) at the beginning and after treatments, and stored at −80 ◦C. Patients did a self-collection at home, following the manufacturer's instructions. They took fecal samples free from urine or toilet water with a spatula, and transferred them into provided tubes (with homogenizer and stabilizing liquid). Samples were kept for one week at room temperature and delivered to the dietician, who froze them (−80 ◦C) until use. The OMNIgene-GUT kit provides a valid method to keep RNA at room temperature [22]. After two months, total RNA was extracted from feces (~50 mg) by dissolving in Trizol reagen<sup>t</sup> (Thermo Fisher). RNA concentration and purity were assessed by the 260/280 nm-ratio using the Nanodrop spectrophotometer (Nirko). Equal amounts of RNA were reverse-transcripted to obtain the cDNA for quantitative-PCR (qPCR), as previously described [23]. The gene expression assays were labelled with Fam fluorophore, whereas the housekeeping gene was labelled by VIC fluorophore. Amplification conditions were: 2 at 50 ◦C, 10 at 95 ◦C and 40 cycles of 15 at 95 ◦C and 1 at 60 ◦C (AB7500 fast y Quant Studio 5; Thermo Fisher). All samples were prepared in triplicate to obtain their threshold cycle (Ct). If deviation for each triplicate were higher than 0.3 cycles, Ct was not considered. The relative expression for each gene was achieved following the model R = 2−ΔΔCt. The primer setup was designed to target the ribosomal RNA genes (16S) of the major bacterial groups present in the mammalian intestinal microbiota, including the Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria, and Verrucomicrobia phyla [24]. To gain an insight into the bacterial composition, we used specific primers for bacterial species (Table 2). The specificity of primers was checked in silico with the "probe match" facility of the Ribosomal Database Project (http://rdp.cme.msu.edu/), and further validated on the BLAST search (NCBI) [25]. The primers were purchased from Thermo Scientific, and stored at −20 ◦C. The reference ranges for intestinal bacteria were calculated as an average of number of gene copies (NGC) from fecal samples of a control population of volunteer patients (Supplementary Materials Figure S1). These subjects (Spanish; 50% females; 45.0 ± 5.0 years-old; *n* = 100) were non-obese; normoglycemic and normolipidemic; and free from known cardiovascular, malignant, and digestive or intestinal diseases. Thus, characteristics of this control group could be compared with those of the study group.
