Spontaneous, non-sponsored, interventional, controlled, randomized, parallel-arm clinical study.

Primary objective To assess the impact of specific dietary approaches (ketogenic diet, Mediterranean diet, or Mediterranean diet supplemented with GABA-producing probiotics, e.g., Bifidobacterium adolescentis PRL2019) on fibromyalgia (FM) severity-particularly pain, sleep quality, anxiety, and depression-using validated questionnaires in female patients with FM and overweight/class I obesity.

Secondary objectives

• Evaluate effects on anthropometric measures, metabolic profile, and body composition.
• Assess changes in gut microbiota composition and metabolomic analysis.
• Measure neurosteroids with positive allosteric activity on GABA-A or NMDA receptors in saliva and/or plasma.
• Exploratory pilot sub-study to quantify brain GABA and glutamate levels through magnetic resonance spectroscopy (MRS) in a subset of patients.

Methods Participants will be recruited from the Rheumatology Unit outpatient clinics of IRCCS Policlinico San Martino Hospital (Genoa). Eligible subjects will be adult women with FM and overweight/obesity, selected through protocol-defined criteria. Enrollment requires written informed consent.

At enrollment (baseline), participants will complete validated questionnaires assessing FM severity (pain, sleep quality, depression, anxiety, bowel function). Data collection will include sociodemographic information, Mediterranean diet adherence through PREDIMED questionnaire, basal metabolic rate, medical and dietary history, smoking status, blood pressure, and recent laboratory results. Stool, blood, and saliva samples will be collected for routine tests, metabolomics, and ELISA-based measurement of neuroactive steroids like cortisol, progesterone, DHEA.

Within 7 days T1, results will be reviewed and eligible participants randomized to one of three groups: hypocaloric ketogenic diet, Mediterranean diet, or Mediterranean diet plus probiotics. Individualized dietary plans will be provided with written and verbal instructions.

Anthropometric measurements (weight, height, BMI, waist circumference, waist-hip ratio) and body composition via bioelectrical impedance analysis BIA will be recorded at T0, T4, T8, and T12, together with biological sample collection.

Pilot MRS sub-study A subset of participants will undergo brain GABA and glutamate assessment through MRI spectroscopy performed on a 3T Siemens - Prisma magnet at the baseline, before dietary treatment and after 4 weeks. Results will be compared with spectra from control subjects screened through medical history to exclude relevant diseases.

Fibromyalgia FM is a heterogeneous and disabling syndrome characterized by multisite pain, sleep disturbances, cognitive dysfunction "fibro fog", and fatigue. The prevalence of FM is approximately 1-5% in the adult population, with a global female-to-male ratio of about 3:1.

The etiopathogenesis of the disease remains unclear; however, accumulating evidence suggests that FM is a central disorder that generates pain through altered processing of non-painful or mildly painful stimuli. Pain in FM is classified as nociplastic, defined as "pain arising from altered nociception despite no clear evidence of actual or threatened tissue damage causing activation of peripheral nociceptors." Recently, a theoretical model of FM pathogenesis has been proposed, based on extensive literature suggesting a role for gamma-aminobutyric acid GABA-the main inhibitory neurotransmitter of the central nervous system-in the pathophysiology of chronic pain conditions such as FM. In particular, the proposed model posits that reduced GABAergic neurotransmission at the thalamocortical level-potentially related to neuroendocrine imbalance leading to altered activity of neurosteroids with positive allosteric modulation of GABA-A receptors or NMDA receptors-may contribute to the development of fibromyalgia pain. This hypothesis requires validation through preclinical and interventional studies.

FM is typically managed using a multimodal approach that includes both nonpharmacological and pharmacological strategies. The most common nonpharmacological interventions include physical therapy, relaxation techniques, psychotherapy, acupuncture, and dietary therapy.

Diet is, in fact, the primary factor capable of influencing the microbiota-gut-brain axis, and dysbiosis has been associated with multiple centrally mediated disorders, including altered nociception and FM. Indeed, microbial metabolites-particularly short-chain fatty acids SCFAs-are able to modulate central neurotransmission by altering, for example, the GABA/glutamate ratio. Furthermore, the prevalence of overweight/obesity reaches 70% in patients with FM, a value substantially higher than in the general population. The high prevalence of overweight/obesity, dysbiosis, and gastrointestinal alterations in FM suggests that dietary therapy and the microbiota-gut-brain axis may play an important role in the pathogenesis of FM.

Although data regarding the relationship between various dietary approaches and FM are limited, the Mediterranean diet has been associated with lower pain intensity, according to an observational study conducted in 186 individuals with FM. A pilot study involving 18 patients also reported the effectiveness of the ketogenic diet not only in promoting weight loss but also in improving symptom severity in obese women with FM. Similarly, evidence suggests that the ketogenic diet may enhance GABA levels and, more broadly, increase the GABA/glutamate ratio.

In this context, in addition to dietary therapy, it may be appropriate to consider supplementation with bacterial strains capable of producing neurotransmitters-such as GABA-that may be deficient in patients with FM and could exert beneficial effects on the central nervous system.

The primary objective of this study is to evaluate the impact of a specific dietary approach-ketogenic diet, Mediterranean diet, or Mediterranean diet supplemented with GABA-producing probiotics like Bifidobacterium adolescentis PRL2019-on the severity of FM, specifically focusing on pain, sleep quality, anxiety, and depression, as assessed using validated questionnaires in female subjects diagnosed with fibromyalgia and mild overweight/obesity.

The secondary objectives are to assess the impact of the dietary intervention on anthropometric measures, metabolic parameters, and body composition of the participants; to evaluate the effect of the dietary intervention on gut microbiota composition through metabolomic analysis; and to measure levels of neurosteroids with positive allosteric activity at GABA-A or NMDA receptors in saliva and/or plasma. Additionally, an exploratory pilot substudy aims to measure cerebral GABA and glutamate levels using magnetic resonance spectroscopy in a small subset of patients.

Participants in this study will be recruited from the outpatient clinics of the Rheumatology Unit at IRCCS Policlinico San Martino Hospital in Genoa. Given the high female prevalence of FM, the study sample will include adult female patients diagnosed with fibromyalgia and mild overweight/obesity.

Each patient enrolled in the study will self-administer, at the Rheumatology Unit, a battery of validated questionnaires designed to accurately assess the severity of FM, with particular focus on the following symptoms: pain, sleep quality, depression, anxiety, and bowel function. Patients will be asked to complete the questionnaires at enrollment/baseline, every four weeks during follow-up visits as per the study protocol, and at the end of the intervention (12 week).

The questionnaires include the Widespread Pain Index WPI and the Symptom Severity Scale SSS, both used in the diagnosis of FM, as well as the Fibromyalgia Impact Questionnaire FIQR. The FIQR assesses patients' quality of life, including physical function, overall disease impact, and symptoms. It is the most widely used questionnaire in research and clinical practice for estimating disease severity, and a minimal clinically important difference has been defined as a 14% reduction in the score.

To further investigate pain, an 11-point Numeric Rating Scale will be administered to assess the average global pain intensity over the past 24 hours. The PainDETECT Questionnaire PD-Q will evaluate the type and characteristics of pain, particularly targeting neuropathic/nociplastic components, especially in chronic pain patients.

Anxiety and depression will be assessed using the Hospital Anxiety and Depression Scale HADS, which includes 7 items for anxiety and 7 for depression, yielding two independent scores: HADS-A and HADS-D. Sleep quality will be assessed using the Pittsburgh Sleep Quality Index PSQI, which evaluates sleep latency, duration, efficiency, and disturbances.

To identify functional gastrointestinal disorders, particularly irritable bowel syndrome IBS, the Rome IV criteria and the Bristol Stool Form Scale, which classifies stool form and consistency, will be applied.

At the T0 visit, all clinical data necessary for study evaluation will be collected, including sociodemographic information, adherence to the Mediterranean diet (assessed via the validated PREDIMED questionnaire), basal metabolic rate calculated using the Mifflin formula, dietary, clinical, familial, and medical history, smoking habits, blood pressure measurement, and the most recent laboratory test results.

Participants will then undergo blood sampling for routine laboratory evaluations as per clinical practice, with a portion of the sample reserved for metabolomic analysis. Metabolites will be extracted from blood samples using methanol and MTBE for untargeted metabolomic profiling, and the MSK-QC-KIT will be added prior to injection into the UHPLC system. Chromatographic separation will be performed under C18 and HILIC conditions. Mass spectrometry data will be acquired using a hybrid quadrupole-Orbitrap Q Exactive Plus in full-scan mode, both in positive and negative ionization. Data will be processed for deconvolution, peak picking, alignment, and compound identification. A procedural blank sample will be used for background subtraction and noise removal during preprocessing. Compound annotation will be performed using MS-FINDER ver. 3.26, followed by bioinformatic analyses for metabolite and pathway enrichment.

Routine laboratory tests will be conducted at the laboratories of Policlinico San Martino, while untargeted metabolomic analysis of the gut microbiota will be performed at the DINOGMI university laboratories IRCCS Giannina Gaslini.

Finally, participants will also be asked to provide saliva samples, in which levels of neuroactive steroids such as cortisol, progesterone, and DHEA will be measured via ELISA analysis. This analysis will be conducted at the DIFAR university laboratories UNIGE.

Within the following seven days, a visit will take place at the same outpatient clinic designated by the Rheumatology Unit, during which the results obtained at T0 will be reviewed with the participant. If no issues are identified related to potential screening failures, participants will proceed with randomization and receive their dietary plan from the dedicated staff at the same clinic, in collaboration with the Dietetics and Clinical Nutrition Unit of the same hospital.

During the T1 visit, participants will be randomly assigned to one of three dietary groups: hypocaloric ketogenic diet, Mediterranean diet, or Mediterranean diet supplemented with probiotics. They will then be provided with the corresponding dietary plan, accompanied by detailed instructions both in writing and through an individual consultation. No adverse effects are expected from the proposed dietary plans, except for the ketogenic diet, where rare side effects are transient and may include nausea, vomiting, temporary lethargy, anorexia, hypoglycemia, dehydration, acidosis, constipation, abdominal bloating, headache, and fatigue.

Anthropometric measurements will be performed, including weight, height, calculation of Body Mass Index BMI, waist circumference, and waist-to-hip ratio. Body composition analysis may be performed using bioelectrical impedance analysis BIA. Anthropometric measurements and biological samples will be collected again during follow-up visits every four weeks and at the end of the 12-week study period.

For the pilot substudy, GABA and glutamate levels will be measured in specific brain regions using magnetic resonance spectroscopy MRS. To this end, a subset of 18 patients-6 per dietary arm-will be selected for this analysis and will sign a specific informed consent form. The procedure will be performed twice: first at T1 depending on scanner availability, prior to the start of the diet, and a second time after 4 weeks.

The spectroscopy results, focusing on GABA and glutamate levels in the patients, will be compared with spectra obtained from six control subjects. Control subjects will be screened using remote and recent medical history to exclude the potential presence of any organ-specific pathologies at the time of assessment by the neuroradiologist. As per routine clinical practice, a pre-examination questionnaire will be administered to evaluate any contraindications, and it will be signed by both the supervising neuroradiologist and the participant. No contrast agent will be administered.

Recruitment of control subjects is necessary because measuring the GABA-glutamate peak in CNS regions is challenging and complex. Establishing baseline GABA and glutamate levels in healthy controls is essential for comparison with FM patients, who may exhibit altered levels. If any pathological findings are suspected during the MRS exam in either controls or FM patients, the neuroradiologist will inform the subjects and recommend a complete morphological MR study, with contrast if necessary.

Magnetic resonance imaging MRI uses radiofrequency waves rather than ionizing radiation, unlike computed tomography, and with appropriate sequences, no specific risks are currently known for participants. Data processing will be performed offline using Gannet software version 3, developed for single-voxel 1H-MRS data analysis to measure GABA and glutamate. Metabolite concentrations will be expressed in institutional units, relative to water, with a correction factor derived from the cerebrospinal fluid fraction in the voxel. The software enables co-registration of the structural image with the voxel and segmentation into the three main tissue types to compute this correction factor. Changes in metabolite concentrations between T0 and T4 will be calculated relative to baseline, and T4 values will be compared with the mean values of the controls.

The MRI scanner used will be a 3T Prisma Siemens, available at the Neuroradiology Unit and routinely used for both clinical and research purposes. The MEGA-PRESS sequence, included in the acquisition protocols, will be used for this type of measurement. Two voxels-30 × 30 × 30 mm³- will be positioned: one in the left thalamus and one in the left parietal somatosensory cortex. These regions were chosen as representative of sensory cortex and thalamic relay structures, reflecting sensory pathways, including pain processing. Additionally, two structural imaging sequences, T1-MPRAGE 3D and FLAIR-3D, will be acquired for data analysis.

Inclusion Criteria

• Adult female patients diagnosed with fibromyalgia, aged ≤ 65 years, with overweight or class I obesity (24.9 < BMI ≤ 34.9 kg/m²).
• Patients capable of providing written informed consent for study participation independently.
• Patients motivated to participate in the study and able to manage the dietary plans autonomously.

Exclusion Criteria

• Lack of signed informed consent.
• Age < 18 or > 65 years.
• Male sex.
• Patients with food allergies or celiac disease, type 1 diabetes mellitus or autoimmune forms of diabetes, or severe pancreatic beta-cell dysfunction; patients treated with SGLT2 inhibitors; those with recent cardiovascular or cerebrovascular events, liver failure, moderate or severe renal failure, respiratory insufficiency, gout episodes, kidney stones, electrolyte imbalances, NYHA class III-IV heart failure, unstable angina, cardiac arrhythmias, or myocardial infarction within the past 12 months; severe depression or other psychiatric disorders; drug, narcotic, or alcohol abuse; frail patients; pregnancy or breastfeeding; patients scheduled for surgery or invasive procedures; rare metabolic disorders like porphyria, carnitine deficiency, carnitine-palmitoyl transferase deficiency, carnitine-acylcarnitine translocase deficiency, pyruvate carboxylase deficiency, or mitochondrial beta-oxidation disorders; patients on diuretic therapy.
• Use of prebiotics and/or probiotics not specified by the study or other dietary supplements within one month prior to enrollment.
• Antibiotic treatment within 3 months prior to recruitment.
• Active infections at the time of recruitment or any chronic gastrointestinal disorder e.g., Crohn's disease.
• Significant eating problems e.g., dysphagia. Additional Exclusion Criteria for MRI
• Implanted metallic devices e.g., pacemakers.
• Head, neck, or body size incompatible with MRI scanner.
• Presence of piercings or severe claustrophobia. Study Sample
• 90 female patients: 30 assigned to the ketogenic diet VLCKD, 30 to the Mediterranean diet MD, and 30 to the Mediterranean diet supplemented with probiotics MDP. A 10% dropout rate is anticipated based on the literature on fibromyalgia treatment and dietary interventions.

Pilot Substudy

• 18 female patients, 6 per dietary group, will be included for the MRS analysis.

Study Duration and Assessments

• Each patient will undergo a 12-week dietary intervention.
• Baseline assessment at T0, randomization and dietary plan delivery at T1.
• Follow-up visits at 4, 8, and 12 weeks T4, T8, T12.
• Optional phone contacts at 2 or 6 weeks T2 or T6 if necessary.

Mediterranean Diet A Mediterranean hypocaloric diet of 1,400 kcal/day will be provided. Macronutrient distribution will follow the Italian Reference Intake Levels for Nutrients and Energy -LARN, 5th edition : 0.8-1.2 g/kg of body weight for protein, 45-60% of calories from carbohydrates, and 20-35% from lipids. Daily caloric allocation will be: 15% at breakfast, 35-40% at lunch, 30-35% at dinner, and the remaining 10% divided into two snacks. Each participant will follow the Mediterranean diet for the entire 12-week intervention, with adjustments as needed to ensure compliance.

Very Low-Calorie Ketogenic Diet VLCKD:

The VLCKD is a low-carbohydrate, reduced-calorie dietary regimen providing adequate protein. Its mechanism is based on ketone production, used as an alternative energy source to glucose. Ketosis occurs when carbohydrate intake is extremely low, forcing the body to mobilize fat for energy. VLCKD has proven effective for rapid weight loss in obese individuals who failed other dietary interventions and is included as a therapeutic option in obesity management guidelines. Recent evidence suggests additional benefits on mood, cognitive function, nociception, and sleep quality.

In this study, participants will receive food kits prepared by SDM (Pronokal Group), providing all meals and vitamin supplements required for nutritional balance and VLCKD compliance. The intervention consists of three phases:

• Phase I - "Attack" during 4 weeks: 800 kcal/day, 1.2 g/kg ideal body weight of protein, 10-15 g lipids, 45-70 g carbohydrates; daily supplementation includes 2 bs/day of UNICOMPLEX multivitamins, 3-4 PronoKal meals, 2 tablespoons of oil, and low-starch vegetables (e.g., chard, broccoli, cauliflower, chicory, rapini, zucchini, valerian).

• Phase II - Transition during 4 weeks: Gradual reintroduction of carbohydrates, increased caloric intake, and reduced PronoKal product use; 1 bs/day of UNICOMPLEX is maintained. Stepwise introduction:

  1. Week 1 - fruit and cereals at breakfast (+150 kcal)
  2. Week 2 - dairy and cereals at lunch (+150 kcal)
  3. Week 3 - legumes (+150 kcal)
  4. Week 4 - increase calories from previously introduced foods (+150 kcal)

• Phase III - Maintenance during 4 weeks: Hypocaloric Mediterranean-style diet of 1,400 kcal/day, completing the 12-week intervention.

A third group of 30 participants will follow a personalized Mediterranean diet, as described above, enriched with GABA-producing probiotics, e.g., Bifidobacterium adolescentis PRL2019 20 CFU/day for 12 weeks.

Sample size For the main study, sample size was estimated based on the FIQ test. Expected differences between baseline and treatment were 12 points for the best-performing intervention, and 8 and 6 points for the other two, with an assumed within-group SD of 7, significance α = 0.05, and power 1-β = 0.8. This resulted in a total n = 81. To account for 10% dropout, 30 patients per arm, total n = 90, will be recruited.

Due to limited scanner availability, the MRI analysis will be a pilot study, common for assessing feasibility without formal power calculations. Three groups, one per intervention arm, of n = 6 patients each will undergo MR measurements at T1 and T4 at the Neuroradiology Unit, Policlinico San Martino. Data will be compared with single-measure MR scans from matched control subjects.

The statistical analysis will be performed using IBM SPSS Statistics version 25 and R version 4.3.3, depending on the statistical requirements. The assumption of normality for each variable will be assessed using the Shapiro-Wilk test. Continuous variables will be reported as median and interquartile range or mean ± standard deviation, depending on whether they follow a normal distribution. Ordinal and nominal variables will be summarized in contingency tables showing frequencies and percentages within the study population.

Comparisons of continuous and ordinal variables between patient groups will be performed using parametric or non-parametric tests of mean differences, depending on group size, or analysis of variance ANOVA. Nominal variables will be analyzed in contingency tables using Pearson's chi-square χ² test or Fisher's exact test. Specifically, for questionnaire outcomes, treatment efficacy will be assessed by evaluating pre-post score differences between groups and their standard deviations to calculate effect size. Statistical significance will be evaluated using ANOVA models parametric or non-parametric, as appropriate.

Metabolites showing significant differences between groups will be identified using one-way ANOVA, followed by Tukey's HSD post-hoc test based on the p-value threshold. Changes in metabolite levels will be visualized using the EnhancedVolcano package version 1.12.0. Differences in cytokine and chemokine levels will be assessed using ANOVA and Student's t-test. A p-value ≤ 0.05 will be considered indicative of a statistically significant difference between groups.

Inclusion Criteria:

• Adult female patients with fibromyalgia, aged ≤ 65 years, who are overweight or obese (BMI 24.9 < ≤34.9 kg/m2);
• Patients able to independently provide their written informed consent to participate in the study;
• Patients motivated to participate in the project and able to independently manage the dietary plans provided for in the study.

Exclusion Criteria:

• Lack of signature on informed consent form
• Age < 18 and > 65 years
• Male gender
• Patients with food allergies or celiac disease, type 1 diabetes mellitus and autoimmune forms of diabetes mellitus or with severe pancreatic beta cell dysfunction, undergoing treatment with SGLT2 inhibitors, with recent cardiovascular or cerebrovascular events, liver failure, moderate or severe renal failure, respiratory failure, episodes of gout, kidney stones, electrolyte disturbances, NYHA class III-IV heart failure, unstable angina, cardiac arrhythmias or myocardial infarction within the last 12 months, severe depression or any other psychiatric illness, drug, narcotic or alcohol abuse, frail patients, pregnancy and/or breastfeeding, patients about to undergo surgery or invasive procedures, rare diseases (porphyria, carnitine deficiency, carnitine palmitoyltransferase, carnitine acylcarnitine translocase and pyruvate carboxylase, mitochondrial beta oxidation disorders), treatment with diuretics.
• Use of prebiotics and/or probiotics not included in the study, or other dietary supplements in the month prior to enrollment
• Antibiotic treatment less than 3 months prior to enrollment
• Infections at the time of enrollment or any chronic gastrointestinal disorder (e.g., Crohn's disease)
• Significant eating problems (e.g., dysphagia).

All measures data as reported in the list of outcomes.

Data will be share upon publication in peer review journals.

Data will be uploaded on open access research repository and identified by a doi.