BackgroundPain accompanying mastitis has gained attention recently as a relevant welfare compromising aspect of disease. Adequate pain recognition and therapy are necessary in the context of a modern and ethically acceptable dairy care. For research purposes mastitis is often induced by intramammary infusion of immunogenic bacterial cell wall components. Lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Staphylococcus aureus are commonly administered to this end. While the immune response to specific immunogenic components has been well characterized, not much is known about their role on the expression of pain indicators. The aim of this study was to trial the effects of an intramammary challenge of LTA or LPS on the degree of pain and discomfort as indicated by both physiological and behavioral variables in cows. The hypothesis was that a similar degree of pain can be identified in LTA as well as in LPS induced mastitis.ResultsOn the challenge day, compared to pre-challenge, total pain index increased for all treatment groups (LPS; LTA and control), the LPS group having significantly higher values than the control group (P = 0.01). Similarly, pain visual analogue scale (VAS) increased significantly in all cows following treatment on the challenge day. Furthermore, compared to baseline, higher VAS were found 3, 4 and 5 h after the challenge in cows of the LPS group (P3h, 4h < 0.001 and P5h = 0.001) and 7 h after the challenge in cows of the LTA group (P7h = 0.002). In the control group, VAS was higher 5 h after the challenge (P5h = 0.001). On the challenge day, udder edema was higher in the LPS than in the control group (P = 0.007). Furthermore, 4 h after the challenge, milk cortisol was significantly higher than at baseline in the LPS group (P < 0.001).ConclusionsWhen administered at equipotent doses targeting a standard somatic cell count increase, intramammary LPS seems to be accompanied by a higher degree of pain and discomfort than LTA, as suggested by the modifications of the outcome variables total pain index, VAS, udder edema and milk cortisol.Electronic supplementary materialThe online version of this article (doi:10.1186/s13028-017-0306-z) contains supplementary material, which is available to authorized users.