Low-FODMAP Diet and Probiotics: Effects on Gut, Behavior, and Microbiota in Children With Autism Spectrum Disorder

This randomized, controlled, parallel-group clinical trial aimed to investigate the effects of a low-FODMAP diet and multi-strain probiotic supplementation on gastrointestinal (GI) symptoms, behavioral problems, and gut microbiota composition in children with Autism Spectrum Disorder (ASD) who presented with concurrent GI complaints.

Children aged 6 to 12 years with clinically diagnosed ASD and GI symptoms were recruited. A total of 16 participants were randomly assigned into two equal groups (n=8 per group). One group received a probiotic supplement containing four bacterial strains daily for 4 weeks, while the second group received the same probiotic supplement in combination with a low-FODMAP diet over the same period. The randomization was stratified and allocation was blinded to the data analysts.

Interventions Probiotic group: A commercially available probiotic blend (containing Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus rhamnosus, and Saccharomyces boulardii) was administered once daily for 4 weeks.

Low-FODMAP + Probiotic group: In addition to the probiotic supplement, participants received individualized nutrition counseling and followed a standardized low-FODMAP diet protocol under dietitian supervision for 4 weeks.

Data Collection and Measures GI symptoms were assessed using the Pediatric Gastrointestinal Symptoms Questionnaire - Rome III Version (QPGS-RIII) and the Bristol Stool Scale (BSS).

Behavioral outcomes were evaluated using the Aberrant Behavior Checklist (ABC), focusing on subscales including irritability, lethargy-social withdrawal, stereotypy, hyperactivity, and inappropriate speech.

Dietary intake was recorded using 3-day food diaries to determine intake of daily FODMAP amount.

Microbiota composition was analyzed from fecal samples using 16S rRNA gene sequencing and LEfSe (Linear Discriminant Analysis Effect Size) for taxonomic biomarker discovery.

Quality Control and Data Validation All participants were monitored weekly to ensure adherence to interventions and assess adverse effects.

Dietary intake data were validated against food portion models and caregiver interviews.

Stool sample processing followed standardized protocols: DNA was extracted, amplified, and sequenced using Illumina platforms.

A pre-defined bioinformatics pipeline was used for quality filtering, taxonomic assignment, and alpha/beta diversity analysis.

Internal consistency checks and double data entry validation were applied to all behavioral and symptom questionnaires.

Statistical Analysis Statistical analysis was performed using SPSS v25 and R. Paired and independent-sample t-tests or Wilcoxon signed-rank tests were applied based on distribution normality. Microbiota diversity indices (Shannon, Simpson, Chao1) and taxonomic differences were compared across groups and time points. Spearman correlation analyses were used to explore associations between microbiota shifts and clinical parameters. A p-value of <0.05 was considered statistically significant.

Inclusion criteria

• Diagnosed with Autism Spectrum Disorder by a specialist at Marmara University Pendik Training and Research Hospital Child and Adolescent Psychiatry Clinic
• Aged between 6 and 12 years
• Diagnosed with functional gastrointestinal symptoms according to Rome IV criteria
• Willing to participate by signing informed consent and assent forms (by both child and parent)

Exclusion criteria

• Following any other dietary intervention at the time of study initiation
• Routine use of prebiotics or probiotics
• Use of antibiotics within the 4 weeks prior to the study
• Presence of active diarrhea due to intestinal infection or similar condition
• Diagnosis of other gastrointestinal diseases besides functional GI symptoms