Eleven Danish Holstein cows were used to examine the effects of quarter health (healthy vs. unhealthy), milking interval (12 vs. 6 h), and sampling time during milking on the concentration of 8 milk constituents [acetone, beta-hydroxybutyrate (BHBA), N-acetyl-beta-D-glucosaminidase (NAGase), somatic cell count (SCC), urea, fat, protein, and lactose]. The selection criterion was that each cow should have 2 or 3 healthy and 1 or 2 unhealthy quarters. Foremilk was collected before attaching the teat cups of the milking machinery, and thereafter, milk samples were collected automatically from each quarter every 45 s during milking. Compared with milk from healthy quarters, milk from unhealthy quarters had a higher concentration of BHBA, NAGase, SCC, and protein during the entire milking, whereas urea was higher in the last part of the milking process. Healthy quarters had a higher content of acetone and lactose during the whole milking, whereas fat was higher in the first part of the milking process. When the cows were milked at the 6-h interval, all milk constituents except lactose and protein were higher during the whole (NAGase, SCC, and urea) or part of the milking (acetone, BHBA, and fat) compared with when cows were milked at the 12-h interval. Lactose was higher in the first part of the milking at the 12-h compared with the 6-h interval, whereas protein was not affected by milking interval. beta-Hydroxybutyrate, NAGase, SCC, and fat increased during the milking process, whereas acetone, urea, protein, and lactose decreased. Foremilk was remarkably different for all constituents, except acetone, and should not be used as a representative milk sample to achieve the true level of a milk constituent. If these milk constituents are to be used in an inline management system, these effects should be taken into account.