Staphylococcus aureus (S. aureus) often causes long-lasting chronic sub-clinical udder infections in dairy cows. To investigate if this can be due to a negative impact of S. aureus on lymphocytes important for the immune defence, alterations in proportions and expression intensity of CD4+, CD8+, WC1+, B and IL-2R+ lymphocytes was studied in blood and milk, as S. aureus mastitis developed from acute clinical to chronic sub-clinical form. Six healthy dairy cows were inoculated with S. aureus in one udder quarter per cow, and one quarter per cow acted as an uninfected control. Blood samples, and milk samples from infected and non-infected quarters were collected before infection and for five weeks after infection. All infected quarters developed acute clinical mastitis, of which five turned into chronic sub-clinical mastitis. In infected quarters, the proportions of all lymphocyte sub-sets, except WC1+ cells, differed in acute phase compared to pre-infection, while the dominant finding in the chronic phase was increased expression intensities per cell. An impact on blood lymphocytes and milk lymphocytes in non-infected quarters also occurred, mainly during the chronic phase. The most prominent finding was the increased proportion and expression of B-lymphocytes in blood, infected and non-infected quarters during chronic sub-clinical mastitis. As S. aureus can invade and survive intracellularly, a preferential stimulation of B-cells, suggesting development of a humoral response, may not be sufficient to eliminate intracellular bacteria, which could explain the persistence of the infection.