Globally, the majority of dairy cows are milked twice a day (TAD); however, in pasture-based production systems, such as in Ireland, the idea of milking once a day (OAD) is being considered for reasons such as improved work-life balance. The immediate effects within a lactation, as well as the multilactation consequences of OAD, compared with TAD milking, require understanding. The objective of this randomized experiment was to compare OAD and TAD milking, over a 3-yr period, by examining the differences in milk production and composition, body weight (BW), body condition score (BCS), dry matter intake (DMI), udder characteristics, locomotion score, and milking time. Over the 3-yr period, 83 cows were enrolled in the experiment; 32, 44, and 48 cows in yr 1, 2, and 3 of the experiment, respectively. Each year, 23% of the herds were primiparous animals, while the remainder were second lactation or greater in parity. All cows were milked in the morning at 0700 h; only cows milked TAD were milked a second time each day at 1600 h. Cows rotationally grazed pastures for the duration of the lactating period and were housed during the nonlactating period. Milking cows OAD reduced cumulative milk yield by 26%, and milk solids yield (kg of fat + kg of protein) by 21%, across the 3 yr of the experiment when compared with cows milked TAD which produced 4,126 and 365 kg/cow, respectively. A contributory factor to the reduced production was a shorter lactation length (9.7 d) of the cows milked OAD compared with TAD (294 d). Milk fat percent of cows milked TAD was similar for all 3 yr of the study (5.05%), whereas milk fat percent of the cows milked OAD increased year on year, with each year being greater than the previous year (5.02%, 5.32%, and 5.70% for yr 1, 2, and 3; respectively). Milk protein percent was greater (+0.19%) for cows milked OAD compared with TAD which was 3.78%. Compared with cows milked TAD, total DMI for cows milked OAD was 22% less at the start of lactation (<167 d), but as the lactation progressed (>167 d) we observed no difference in DMI between treatments. Similar to the literature, milking cows OAD significantly increased average somatic cell score, both during (+16%) and at the end of lactation (+19%), compared with milking cows TAD which were 4.69 and 4.79, respectively. We detected positive aspects associated with OAD milking such as greater BW, BCS, and fertility performance. Milking OAD reduced both milking time per cow per day (reductions ranged from 34% in the first 4 mo of lactation to 43% during mo 5-9 of lactation) and milking time per liter of milk (-3.5 s/L) throughout lactation, leading to less labor inputs on-farm which can have positive implications for farmer work-life balance. The significant time saving and potential savings in costs (e.g., electricity) need to be considered in conjunction with the milk production reduction when considering OAD milking for the entire lactation., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)