Mastitis is the most prevalent disease in dairy cows, however, alleviation of pain is rare. This study aimed to assess the relationships between behavioral signs of pain and physiological responses to mammary inflammation, and provide evidence of the need for mastitis pain relief. Lipopolysaccharide (LPS) induced mastitis was used to introduce a controlled amount and type of non-replicating irritant to elicit symptoms of clinical mastitis, along with rapid elimination and recovery; typically within 24-36 h. Ethics approval was received. Twenty Holstein cows were assigned to five blocks of four cows grouped by parity and stage of lactation. Cows were individually housed in tie-stalls and continuously video-monitored for the duration of the study, which consisted of a 1-week pre-experimental period followed by a 4-day experimental period. Two cows within a block were randomly selected to receive either intramammary infusion of 25 µg of sterile LPS or intramammary infusion of sterile saline. Blood samples, milk samples, and rectal temperatures were collected five times on the day of challenge and then once daily for the next three days. Behavior was observed continuously for 24 h after infusion. Data were analysed by ANOVA. LPS treated cows had higher (P=0.05) mean (±SE) rectal temperatures (41.4±0.2 vs. 38.4±0.2 °C), concentrations of milk amyloid (3.4±0.3 vs. 1.4±0.2 µg/ml) and serum cortisol (63.0±6.0 vs. 28.3±2.9 µg/dl) in the 24 h after infusion compared with saline treated cows. LPS treated cows spent less time in the 24 h after infusion eating (16.9±0.8 vs. 21.0±1.2%), ruminating (35.8±2.3 vs. 39.8±1.5%), and lying down (40.7±4.0 vs. 47.9±3.4%) compared with saline infused cows. Using LPS infusion to model clinical mastitis, the results of this study suggest that the behavior of cows is significantly affected as a result of mammary inflammation, and that treatment of both the animal's infection and pain must be considered.