Nutritional interventions hold great promise for managing metabolic dysregulation, significantly impacting the gut microbiome (GM) and weight status. The GM produces bioactive metabolites from gastrointestinal contents, a process influenced by nutrient composition, feeding frequency, and meal timing. Intermittent fasting (IF) and caloric restriction (CR) affect GM stability and diversity by regulating growth and diversity in response to nutrient availability. Protein pacing (P), characterized by evenly spaced, protein-rich meals, further modulates metabolizable energy. This study compares two low-calorie diets matched for weekly energy intake and expenditure: a heart-healthy CR diet aligned with US dietary recommendations and a calorie-restricted IF-P diet. The hypothesis is that IF-P will favorably influence GM and metabolome to a greater extent than CR. This investigation uses data from a previously published randomized controlled trial (NCT04327141) with additional analysis of high and low weight loss responders to the IF-P diet, including a longitudinal year-long follow-up of a high-responder individual.

Previous research established the impact of dietary interventions on the gut microbiome and weight management. Studies have shown that caloric restriction and intermittent fasting influence the gut microbiome, potentially affecting body weight and metabolic health. The role of dietary protein and its interaction with caloric restriction on the gut microbiome has been investigated preclinically in mice, revealing anti-obesity effects. Furthermore, previous research characterized dietary approaches of calorie-restricted IF-P and P alone, revealing beneficial changes in body composition and cardiometabolic health. However, a direct comparison of IF-P with a calorie-matched CR diet in humans, with a focus on GM and metabolomic changes, is lacking. This study aims to fill this gap.

Forty-one overweight or obese individuals (BMI > 27.5 kg/m², % body fat > 30%) were randomly assigned to either IF-P or CR for 8 weeks. The IF-P diet consisted of 35% carbohydrate, 30% fat, and 35% protein, with a weekly extended fasting period (36–60 h) at 350–550 kcals per day. The CR diet followed US dietary recommendations (41% carbohydrate, 38% fat, and 21% protein) with approximately 1200–1500 kcals per day. Both groups reduced their caloric intake by approximately 40% from baseline. Gut symptomatology was assessed using the GI Symptom Rating Scale (GSRS). 16S rRNA gene sequencing and shotgun metagenomics were used to characterize GM composition, while targeted LC-MS/MS and untargeted LC-MS were employed for plasma and fecal metabolomic profiling, respectively. GC-MS was used to analyze fecal short-chain fatty acids (SCFAs). Statistical analyses included linear mixed-effects models, permutational analysis of variance (PERMANOVA), MaAsLin2, and multi-omics factor analysis (MOFA). Subgroup analysis of high and low weight loss responders to IF-P and a year-long case study of a high responder were also performed.

The IF-P group exhibited significantly greater weight loss (-8.81 ± 0.71%) than the CR group (-5.40 ± 0.67%), accompanied by reductions in total, abdominal, and visceral fat mass, and increased fat-free mass. IF-P also significantly reduced gut symptoms compared to CR. While both groups showed changes in GM alpha diversity over time, the IF-P group showed greater intra-individual divergence in GM composition. IF-P significantly increased abundances of *Christensenellaceae*, *Rikenellaceae*, and *Marvinbryantia*, all associated with favorable metabolic profiles. Conversely, it decreased several butyrate-producing taxa. The IF-P group also exhibited significant increases in circulating IL-4, IL-6, IL-8, and IL-13, cytokines linked to lipolysis and weight loss. Plasma metabolomic analysis revealed significant differences between IF-P and CR groups for 15 metabolites, including increases in malonic acid and acetylcarnitine in IF-P. MOFA analysis identified latent factors capturing distinct microbial and metabolomic signatures associated with IF-P and CR. Subgroup analysis of high versus low weight loss responders within the IF-P group revealed further differences in GM composition and fecal metabolic profiles, highlighting the influence of GM on weight loss responsiveness. The longitudinal case study showed long-term GM remodeling and metabolic changes in response to sustained weight loss on an IF-P regimen.

The study's findings demonstrate the distinct effects of IF-P on gut health and metabolism compared to CR. The superior weight loss and gut microbiome remodeling observed in the IF-P group may be attributed to a combination of factors, including reduced caloric intake, intermittent fasting, increased protein intake, and the type and timing of nutrient consumption. The observed increases in specific microbial taxa and cytokines suggest a complex interplay between diet, gut microbiota, and host metabolism. The differences in weight loss responsiveness between individuals within the IF-P group further emphasize the importance of personalized approaches to dietary interventions. The results of the year-long case study support the hypothesis that long-term GM changes contribute to sustained weight loss.

This study shows that IF-P is superior to CR for weight loss and improves several gut health measures. The observed changes in the gut microbiome and metabolome are associated with improved body composition and metabolic markers. Further research should investigate the mechanisms behind these observations, ideally with larger samples sizes and longer study durations. Personalized approaches to dietary interventions should consider these findings to optimize weight management and metabolic health outcomes. Future studies should also explore the role of the upper GI tract microbiome in these interventions.

The use of fecal samples may not fully represent the entire GM. The sample size, although sufficient for the primary outcomes of the parent study, may limit the statistical power to detect subtle differences. The 8-week study duration may not capture long-term effects; however, this was partly addressed by the year-long case study. The use of both whole foods and meal replacements in the IF-P group introduces complexity, making it difficult to isolate the effects of each. Finally, self-reported dietary intake relies on participants accurately tracking their food and supplement consumption.