Once-daily milking (ODM) is known to induce udder inflammation, and could lead to a higher mastitis incidence due to increased milk leakage and extended milk stasis that could promote penetration and multiplication of pathogens. As a fact, ODM increases milk somatic cell counts (SCC) with or without an increased number of clinical mastitis. However, bacteriological analyses are often lacking to describe udder inflammation. Our study aimed to describe the effect of initial health status of the udder on the inflammatory response observed when implementing ODM. The trial used 63 cross-bred Holstein × Normande dairy cows at 78±5 days in milk and comprised a 1-wk control period (cows milked twice daily) and a 3-wk period of ODM (d1, 1st d of ODM). The udder and animal inflammatory status were assessed by measuring SCC 4 d/wk in whole-udder milk, interleukins (IL) 6 and 8 in quarter milk, and haptoglobin in plasma on d0, 1, 2, 3 and 14. The infectious status of quarters was determined at d0, 3 and 14 by milk bacteriological analysis and identification of minor and major pathogens by 16S DNA sequencing. Four initial infectious and inflammatory udder’s classes (57 of the 63 cows) were determined: no infected and inflamed quarters (n=13), no infected quarters but at least one inflamed quarter (n=15), at least one quarter inflamed and infected with a minor pathogen (n=14), and at least one quarter inflamed and infected with a major pathogen (n=15). Milk yield losses due to ODM were higher for cows with inflamed udders compared to non-inflamed udders (25-27% vs 17%, respectively). Implementing ODM increased SCC and IL8. The increase in IL8 was higher for cows infected with a major pathogen. Cows with no infected but inflamed quarters contrasted by showing only a slight increase in SCC with no change in IL8 and haptoglobin. Inflammatory and milk yield responses to ODM thus varied according to the initial udder health status.