Antibody levels against Ostertagia ostertagi in the milk are a promising parameter to identify dairy cows or herds with production losses due to gastrointestinal nematodes. However, milk antibody levels can be influenced by udder-related factors. In this study, the effect of an experimentally induced mastitis on the test results (ODR) of a milk O. ostertagi ELISA was investigated. Twenty-five cows that were naturally infected with gastrointestinal nematodes, were inoculated in their left udder quarters with Escherichia coli P4:O32 and quarter milk samples were collected at several intervals from 24 h before until 144 h after experimental infection. The effect of the contribution of milk from one or more infected quarters on the bulk tank milk ODR was estimated, based on a titration experiment. The mean O. ostertagi antibody levels of the infected udder quarters were significantly (P < 0.001) higher than those of the uninfected udder quarters at each sampling time post-infection. The largest difference was observed at 24 h post-infection with a mean difference of 0.251 ODR (95%CI: 0.172; 0.330). There was also a significant increase (P < 0.001) in total IgG levels with the largest difference being observed at 24 h post-infection. Highly significant (P < 0.005) correlation coefficients were observed between O. ostertagi ODR, total IgG ODR, Na+ and Cl- ion concentration and log transformed somatic cell counts at 24 h post-infection. The results demonstrate that an acute mastitis causes a flow of specific and non-specific antibodies from serum to milk with a subsequent increase in the O. ostertagi ODR values. The effect of the contribution of milk from infected quarters on the bulk tank milk O. ostertagi ODR was estimated to be minor if the relative number of infected quarters is small (< 3%).