In both animals and humans, psychological stress had a deleterious impact on the intestinal barrier function. During public speaking, researchers sought to determine how Lactobacillus rhamnosus CNCM I-3690 affected intestinal permeability and stress markers. With 46 students in each treatment group, healthy students were randomized to drink L. rhamnosus-containing (test) or acidified (placebo) milk twice daily for 4 weeks. A 2 h urinary lactulose–mannitol ratio (LMR, primary outcome), fractional excretion of lactulose (FEL), and mannitol excretion were used to determine small intestine permeability fractional excretion of mannitol (FEM). Salivary cortisol was measured and the State-Trait Anxiety Inventory (STAI) and Perceived Stress Scores (PSS). There were no differences between treatments for LMR (p=.71), FEL, or FEM. Within-treatment analyses revealed comparable LMR and FEL, but a stress-induced rise in FEM with the placebo (p<.05) but not the test product. Despite a similar increase in salivary cortisol, the test product considerably reduced the stress-induced rise in STAI (p=.01). PSS was also found to have a stress-preventative effect, which was more prominent in subjects with high stress-induced cortisol (p=.01). While the impact of the test product on subjective stress was mediated by changes in FEM, the effect of the test product on FEM was not mediated by changes in FEM. There were no significant side effects. Finally, researchers showed that L. rhamnosus CNCM I-3690 reduced stress-induced mannitol hyperpermeability. Subjective but not objective stress markers were lower with L. rhamnosus than placebo, implying anxiolytic effects independent of barrier stabilization and promising for stress reduction in both health and disease.