Hypothesis\n\nWe hypothesize that the anxiolytic effect of Lactobacillus plantarum supplementation is mediated primarily through vagal‑dependent increases in heart‑rate variability (HRV) that precede measurable reductions in peripheral IL‑6, and that this temporal sequence can be captured with twice‑daily short‑term HRV recordings.\n\n### Mechanistic rationale\n\n- L. plantarum reduces gut‑derived TNF‑α and IFN‑γ, lowering intestinal permeability and limiting translocation of bacterial LPS into circulation (source).\n- Decreased LPS signaling attenuates activation of vagal afferents via the nodose ganglion, shifting the balance toward efferent vagal output, which enhances HRV (high‑frequency power) (source).\n- Increased vagal efferent activity stimulates the cholinergic anti‑inflammatory pathway, suppressing splenic IL‑6 production (source).\n- Thus, an early rise in HRV reflects heightened vagal tone that drives downstream cytokine modulation, positioning HRV as an early mechanistic biomarker.\n\n### Testable predictions\n\n1. In a randomized, double‑blind, placebo‑controlled trial of adults with mild anxiety, L. plantarum (≥1 × 10¹⁰ CFU/day) will produce a statistically significant increase in 5‑minute resting HRV (RMSSD) within 48 h of the first dose, detectable before any change in plasma IL‑6.\n2. The magnitude of HRV increase at 48 h will mediate (account for ≥40 % of) the reduction in State‑Trait Anxiety Inventory (STAI) scores at week 4, as tested by causal mediation analysis.\n3. Blocking vagal signaling (via transcutaneous auricular vagus nerve stimulation sham or low‑dose atropine) will abolish both the HRV rise and the anxiolytic effect, confirming necessity of the vagal route.\n\n### Experimental design\n\n- Participants: n = 120, aged 18‑45, baseline STAI ≥ 40, HRV < 30 ms RMSSD.\n- Arms: (A) L. plantarum, (B) placebo, (C) L. plantarum + atropine (low dose to transiently inhibit vagal efferents).\n- Procedures: Baseline HRV (5‑min supine, ECG) and blood draw (IL‑6, TNF‑α). HRV measured twice daily (morning & evening) for 14 days, then weekly. Cytokines assayed at days 0, 2, 7, 14, 28.\n- Analysis: Mixed‑effects models for HRV over time; mediation analysis (HRV Δ at 48 h → STAI Δ at week 4); ANOVA for cytokine trajectories.\n\n### Falsifiability\n\nIf HRV does not rise significantly earlier than IL‑6 changes, or if vagal blockade does not attenuate the HRV‑anxiety link, the hypothesis is refuted, indicating that immune pathways dominate or that HRV is merely a correlative marker.\n\n### Potential impact\n\nConfirming HRV as an early, vagally mediated readout would streamline strain‑specific probiotic trials, enable personalized dosing based on autonomic response, and clarify the gut‑brain axis hierarchy.