Objectives: Little information is available on the properties of fermented milk formula intended to healthy infants. This study analyzes the effect of long-term ingestion of a heat-treated, fermented milk formula on the development of oral tolerance or systemic immune response to soluble antigens in mice. Materials and Methods: The C3H/HeN mice, fed with a heat-treated fermented (Bifidobacterium breve C50 and Streptococcus thermophilus 065) infant formula (htFF) or a matched control diet (control), were immunized with ovalbumin (OVA) with or without gavage of 20 mg OVA to induce tolerance or immunity, respectively. Systemic and local anti-OVA immune responses and intestinal barrier function were measured after 5 to 6 weeks. Results: Oral tolerance to OVA developed similarly in htFF- and control-fed mice, attested to by the downregulation of OVA-specific immunoglobulin (Ig) G and IgE after oral OVA administration. In contrast, immunization with OVA led to significantly higher titers in htFF-fed mice than in control-fed mice (log2 IgG titers, 16.45 +/- 1.24 and 15.46 +/- 0.79, respectively; P = 0.012). Jejunal interferon gamma, interleukin 12p40 and interleukin 10 expressions were significantly higher in tolerized mice fed with htFF compared with those fed with the control diet. Mucosal to serosal intact horseradish peroxidase fluxes were lower in htFF-fed mice than in control-fed mice (39 +/- 8 and 118 +/- 38 ng/h.cm(2), respectively; P < 0.0001), indicating that the htFF diet reinforces intestinal barrier capacity to macromolecules. Conclusions: In mice, htFF strengthens intestinal barrier and enhances systemic immune responses to antigens without interfering with the development of oral tolerance, suggesting a potential beneficial effect in host defence and vaccination.