Cows are often shown at dairy shows with overfilled udders to achieve a better show placing. However, it is unclear to what degree "over-bagging" affects the health and well-being of show cows. The goal of this study was to assess the effect of a single prolonged milking interval (PMI) of 24h on the measurable signs of health and well-being in dairy cows in early and mid-lactation and to assess the effect of a nonsteroidal anti-inflammatory drug (NSAID) on well-being during a PMI. Fifteen Holstein cows were studied in early lactation (89.5±2.7d in milk) and were given an NSAID or physiological saline in a crossover design. Ten cows were studied again in mid-lactation (151.6±4.0d in milk). Data on clinical signs of cows' health, behavior, and well-being were collected at 1 or 2h intervals before and during a PMI of 24h. Data from the last 6h of a 12h milking interval were compared with the last 6h of the PMI. Compared with that of a cow in the last 6h of a 12-h milking interval, the behavior of cows in early lactation (saline group) changed during the last 6h of the PMI: we observed decreased eating time (22.4 vs. 16.2min/h), increased ruminating time (13.3 vs. 25.0min/h), and increased hind limb abduction while walking (score 41.7 vs. 62.6) and standing (31.2 vs. 38.9cm). Udder firmness was increased (2.9 vs. 4.5kg) during this period and more weight was placed on the hind limbs (46.4 vs. 47.0%). We also found pathological signs at the end of the PMI: all cows showed milk leaking, and 10 of 15 cows developed edema in the subcutaneous udder tissue. Somatic cell count was significantly increased from 12h to 72h after the PMI. Administration of an NSAID had no influence on measured variables, except that the occurrence of edema was not significantly increased during PMI in the flunixin group (10 of 15 and 6 of 15 cows for the saline and flunixin groups, respectively). In the cows in mid-lactation, different variables were not significantly changed in the PMI compared with baseline values (e.g., eating and ruminating time, occurrence of edema, and abduction). We conclude that the cows' health and well-being were compromised by a single PMI of 24h, because their behavior changed and pathological signs were recorded. Administration of an NSAID had a slight effect on cows' well-being during a PMI. The stage of lactation had more effect on the cows' health and well-being, because fewer variables were changed in mid-lactation.