Lipopolysaccharides (LPS) induce acute mastitis in dairy cows. A single quarter exposed to LPS will exhibit a local response, but neighboring glands may also show a systemic response. Our objective was to characterize the early responses of milk components to intramammary LPS challenge, aiming to distinguish between local and systemic effects. Ten multiparous cows, >70 DIM, were blocked based on milk yield, parity, and DIM. One cow of each block (T) received an infusion of 50 µg LPS in 10 mL of saline in both front and rear quarters of a randomly selected half-udder (TL); the contralateral quarters received 10mL saline (TS). In parallel, the other cow within block (C) received either 10 mL of saline (CS) or no infusion (CN) into respective half-udders. Infusions were given immediately after morning milking (0h). After sanitizing and stripping teats, samples of foremilk (~30 mL) were taken before twice-daily quarter-milking (−24, 0, 12, 24 h relative to infusion) from front quarters only. Additional foremilk samples were collected at 3 and 6 h. Milk composition was similar between all quarters before infusions. SCC and protein content indicated a local response to LPS: SCC was higher in TL milk compared with TS by 6 h (6.48 vs 5.25 log10 cells/mL, P < 0.001) and remained elevated at 24 h (P < 0.001); protein was transiently higher in TL compared with TS (3h; 3.1 vs 2.9%, P < 0.05). Systemic effects were observed for fat content and milk urea nitrogen (MUN): in both TL and TS, fat was lower than in CS and CN (3 h: 3.3, 3.7 vs 6.7, 6.5%, P < 0.01; 6 h: 2.9, 3.2 vs 4.7, 4.8%, P < 0.05) and MUN was lower (12 h: 9.3, 8.8 vs 12.9, 12.3 mg/dL, P < 0.05), respectively. Both local and systemic effects were observed for lactose content: at 6 and 24 h, lactose in TL was lower than in TS (6 h: 3.8 vs 4.2%, P < 0.05; 24 h: 2.9 vs 3.5%, P < 0.01), with both lower than C quarters by 12 h (P < 0.01). In summary, intramammary LPS rapidly altered milk composition with significant changes in T glands by 3 h. Analyzing composition of foremilk revealed distinct local or systemic effects of LPS.