Hypothesis

Individuals with low baseline heart‑rate variability (HRV) and reduced fecal Faecalibacterium will exhibit a significantly greater reduction in anxiety symptoms after a 14‑day supplementation with Lactobacillus rhamnosus JB-1 compared with individuals exhibiting high baseline HRV, because low vagal tone creates a permissive state for vagally mediated GABAergic modulation.

Rationale

• L. rhamnosus JB-1 exerts anxiolytic effects in rodents through vagal afferent signaling that modulates central GABA_A receptors; vagotomy abolishes this effect [2].
• Low HRV reflects diminished vagal tone and is associated with gut dysbiosis (reduced Faecalibacterium, increased Prevotella) and heightened anxiety/depression risk [4].
• When vagal tone is low, the autonomic nervous system operates farther from its homeostatic set‑point, increasing the gain of vagal afferent pathways; thus, an identical probiotic‑induced vagal signal should produce a larger downstream effect on GABAergic tone in low‑HRV individuals.
• This predicts an interaction: baseline HRV moderates the probiotic‑anxiety relationship, independent of absolute changes in microbiota composition.

Predictions

1. Primary – The change in State‑Trait Anxiety Inventory (STAI) score from baseline to day 14 will be negatively correlated with baseline HRV (r < 0) and positively correlated with baseline Faecalibacterium abundance (r > 0).
2. Secondary – Responders (defined as ≥ 30 % reduction in STAI) will show a significant increase in time‑domain HRV (RMSSD) post‑intervention, whereas non‑responders will not.
3. Exploratory – Plasma GABA levels will rise more in low‑HRV responders, supporting a vagal‑GABA mechanistic link.

Experimental Design

• Design: Double‑blind, placebo‑controlled, crossover trial with a 2‑week washout.
• Participants: n = 60 healthy adults screened for baseline HRV (5‑minute resting ECG) and stratified into low (< 30 ms RMSSD) and high (> 45 ms RMSSD) groups.
• Intervention: 1 × 10^9 CFU L. rhamnosus JB-1 daily vs. matched placebo for 14 days.
• Outcomes: STAI, HRV (time‑ and frequency‑domain), fecal 16S rRNA sequencing (focus on Faecalibacterium and Prevotella), plasma GABA, and cytokines (IL‑6, TNF‑α).
• Analysis: Mixed‑effects model with fixed effects for treatment, baseline HRV group, and their interaction; random intercept for participant.

Potential Outcomes and Falsifiability

• If supported: A significant treatment × HRV interaction (p < 0.05) confirming that low‑HRV individuals derive greater anxiety benefit, coupled with increased HRV and GABA in responders, would validate the hypothesis that vagal tone gates probiotic efficacy.
• If falsified: No interaction, or a reversed pattern (high‑HRV responders benefit more), would refute the proposed gain‑of‑function mechanism and suggest that other pathways (e.g., direct GABA production) dominate in humans.

This hypothesis is directly testable, extends the existing preclinical work by incorporating individual autonomic state as a moderator, and offers a clear mechanistic bridge between vagal tone, microbial signaling, and anxiety outcomes.