This pilot dietary intervention study aimed to investigate whether short-term consumption of apples influences the human faecal metabolomic profile. Apples are a commonly consumed source of dietary fibre, particularly pectin, which undergoes microbial fermentation in the colon and may lead to measurable metabolic changes. Healthy adult volunteers were instructed to consume three apples per day for three consecutive days under free-living conditions. Faecal samples were collected before and after the intervention and analysed using proton nuclear magnetic resonance (¹H NMR) spectroscopy to assess changes in metabolite concentrations. The study sought to explore whether metabolomic profiling can detect functional microbial responses to increased fruit intake and provide preliminary insight into the metabolic impact of short-term dietary fibre exposure.

This study was designed as a single-arm exploratory dietary intervention to evaluate short-term metabolic responses to increased intake of fruit-derived dietary fibre. Participants were instructed to consume three fresh Gala apples per day for three consecutive days while otherwise maintaining their habitual diet and lifestyle. The intervention was conducted under free-living conditions to reflect realistic dietary behaviour.

Faecal samples were collected immediately before the intervention and after completion of the three-day exposure period. Samples were processed using standardized laboratory protocols to obtain aqueous extracts suitable for metabolomic analysis. Proton nuclear magnetic resonance (¹H NMR) spectroscopy was employed to generate quantitative metabolic profiles, enabling detection of metabolites associated with microbial fermentation and host-microbe metabolic interactions.

Metabolite concentrations were quantified using targeted profiling approaches, and both multivariate and univariate statistical analyses were applied to assess intervention-related changes in the faecal metabolome. The study was intended to provide mechanistic insight into how short-term consumption of a commonly consumed fruit may influence gut microbial metabolic activity, and to evaluate the feasibility of metabolomics-based assessment of dietary exposures in small-scale human intervention studies.

Inclusion Criteria:

• Healthy adults aged 18-30 years
• Able and willing to consume apples daily during the intervention period
• Willing to provide stool samples before and after the intervention
• Not following a specific prescribed diet
• Physically able to maintain usual daily activities during the study
• Able to provide written informed consent

Exclusion Criteria:

• Diagnosed gastrointestinal, metabolic, or chronic systemic disease
• Use of antibiotics, probiotics, or regular medication affecting digestion in the period prior to enrolment
• Known allergy or intolerance to apples or other fruits
• Current participation in another dietary or clinical study
• Following a medically prescribed or restrictive diet
• Pregnancy or breastfeeding

Individual-level participant data will not be publicly shared due to privacy considerations and ethical restrictions. De-identified summary data supporting the study findings may be available from the investigators upon reasonable request.

Due to the nature of the dietary intervention, neither participants nor investigators were blinded to the intervention. All participants received the same exposure, and outcome assessment was based on objective metabolomic measurements.