**2. Methodology**

#### *2.1. Subjects of Study*

A pilot prospective study (NCT03872245) was completed in the Obesity Unit of Garcilaso Clinic (Madrid, Spain). The inclusion criteria were adult patients with a body mass index (BMI) > 30 kg/m2, with previous failure in dietary treatment. The exclusion criteria were (i) untreated endocrine diseases causing obesity, (ii) portable electric devices, (iii) diagnosis of previous cardiovascular events (acute myocardial infarction or coronary syndrome, heart failure) or cancer, and (iv) earlier treatment with hormone, prebiotics, probiotics, or nutritional supplements. In a previous study, we had observed that PENS of dermatome T6 (PENS) associated with the hypocaloric (1200 Kcal) diet produced a significantly greater weight loss (BMI = −5.1 kg/m2) than only PENS (BMI = −1.4 kg/m2) or the isolated hypocaloric diet (BMI = −2.0 kg/m2) [9]. Moreover, data from the literature have shown that single or multistrain probiotics alone produced minimal changes in body weight (BMI = −0.36 and −0.15 kg/m2, respectively) and in glycemic/lipidemic factors [19]. Therefore, we have now treated

obese subjects, who previously were unsuccessfully treated only with the hypocaloric diet, with PENS with or without probiotics under the same diet in order to observe the potential differences in weight loss, associated cardiovascular factors (i.e., blood pressure, glycemia, and lipidemia), and microbiota. Thus, patients (*n* = 20) were randomized into two groups for anti-obesity interventions; PENS in conjunction with a hypocaloric diet (*n* = 10) (PENS-Diet), and the same strategy plus an administration of probiotics (*n* = 10) (PENS-Diet + probiotics). We followed a simple randomization using a random number table. All patients signed an informed consent for inclusion in the study and the use of clinical data for this research project. The Ethical Committee of Clinical Research (Medicine, Esthetic and longevity Foundation) approved this investigation (ref.: Garcilas-19-3; Feb 2019). The work was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). All participants finished the study.

#### *2.2. Percutaneous Electrical Stimulation (PENS)*

The PENS of dermatome T6 was performed as previously described [10] by using the Urgent PC 200 Neuromodulation System® (Uroplasty, Minnetonka, MN, USA). Patients were placed in a supine position without anesthesia, and PENS was delivered by a needle electrode inserted in the left upper quadrant along the medio-clavicular line, at two centimeters below the ribcage, at a 90◦ angle towards the abdominal wall, and at 0.5–1 cm of depth. Successful insertion was confirmed by the feeling of electric movement at least 5 cm beyond the dermatome territory. The PENS was undertaken at a frequency of 20 Hz at the highest amplify (0–20 mA) without causing any pain. The participants underwent one 30-min session every week for ten consecutive weeks.

#### *2.3. Hypocaloric Diet, Exercise, and Probiotics Administration*

A 1200 Kcal/day diet was uniformly prescribed during PENS interventions in both groups of patients, as we previously published. The diet followed a Mediterranean style (carbohydrates 51%, proteins 23% and fat 26%) with a high intake of fruit and vegetables, a moderate intake of meats, and olive oil as the main source of fat [20]. Briefly, patients were recommended to take skimmed milk (200 mL) or natural yogur<sup>t</sup> and bread (200 g) as breakfast, 100 g of fruit (e.g., apple, pear) mid-morning, and 200 g of vegetables (e.g., spinach, lettuce, cauliflower) or pasta soup, fish (120 g) or chicken (100 g), and fruit (100 g), at lunch and dinner. Olive oil (30 cc) could be also taken as a complement, and skimmed milk (200 mL) with coffee or tea as a snack. A record of food intake was applied along the study. The intake of alcohol and nutritional supplements was not allowed during the study. Moreover, patients received instructions for regular exercise practice (1 h of brisk walking/day), following a counselling protocol against obesity in patients under 50 years [21]. Brisk walking consisted of a moderate-intensity exercise of walking to a minimum speed of 100 steps per minute (about 4.8 km/h). Since obese patients have many difficulties in adhering to nutritional advice and exercise recommendations, we did a weekly follow-up of food intake and exercise practice. Our dietician phoned all the patients to remind them of the need to stick to these recommendations. The dietician wrote down the daily intake of food and the time/speed of brisk walking. At the end of the study (10 weeks), the dietician confirmed a full adherence rate to the Mediterranean diet and daily exercise. In a previous work, we described a 98% and 94% diet compliance in patients undergoing PENS or PENS-Diet interventions, respectively [9]. The reduction of appetite induced by PENS and the short length of the study could facilitate this high adherence. Some patients additionally received two tablets per day of probiotics Adomelle® (4th generation technology, Bromatech, Italy) during the ten weeks of treatment. The composition of Adomelle® was *Lactobacillus plantarum LP115* (<1 × 10<sup>9</sup> colony forming units; CFU)*, Bifidobacterium brevis B3* (<1 × 10<sup>9</sup> CFU), and *Lactobacillus acidophilus LA14* (1 × 10<sup>9</sup> CFU). The probiotics were given after meals with drinking water and did not alter the food intake. Participants were also compliant with probiotics intake.
