Background and aims The immune mechanisms by which probiotics reduce susceptibility to upper respiratory tract illness is uncertain. The aim of this study was to examine purported cell-mediated immune mechanisms that might explain the reduction in respiratory illness observed following daily supplementation with Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04) and a combined Lactobacillus acidophilus NCFM & B. animalis subsp. lactis BI-07 (NCFM & Bi-07). Methods A cohort of 144 healthy physically active individuals were allocated to daily supplementation consumed as a beverage with Bl-04 (n = 46) supplemented at a dosage of 2.0 נ109 colony forming units (cfu) per day, NCFM & Bi-07 (n = 47) at a dosage of 5.0 נ109 CFU per day each, or a placebo (n = 51) over 150 d. Markers included plasma cytokines, metalloproteinases and neurotrophins, peripheral blood leucocyte numbers, antibody-dependent and antibody-independent NK cell activity (NKCA), and peripheral blood mononuclear cell (PBMC) phagocytosis. Results A total of 125 subjects were included in the final analysis. No significant effects were observed on cytokines, on white cell differentials, NKCA or PBMC phagocytosis from pre- to post-supplementation. The biomarkers that increased significantly from pre- to post-supplementation were the concentration of plasma macrophage inflammatory protein (MIP)-1d which was higher in the Bl-04 than placebo group (Bl-04 25% ᠱ1%, placebo -3.3% ᠹ.4%; mean ᠓D, P = 0.003) while the concentration of plasma matrix metallo-proteinase (MMP)-1 decreased by 11% ᠱ6% in the NCFM & Bi-07 group and increased by 21% ᠱ7% in the placebo group, which was a significant 26% difference (8-41%; P = 0.02). Conclusion Probiotic supplementation had little effect on parameters of the innate immune system. Mechanisms explaining the beneficial effect of Bl-04 or NCFM & Bi-07 supplementation on respiratory illness remain unclear.