The objective was to determine the effect of once-daily milking (ODM) and omitting one evening milking each week (13TWM), in late lactation on milk production, composition and processability. Seventy-two cows were assigned to three treatments (ODM, 13TWM and twice-daily milking [TDM]) from 4 October to 12 December. Cows were on average 218 d into lactation at the start of the trial, and all cows were managed similarly throughout the trial. Milk yields and gross milk composition of cows on all treatments were measured, and milk samples for detailed compositional and processability analysis were collected from TDM and ODM treatments at two consecutive milkings and at one milking each week, respectively. Milk yield was significantly reduced (P < 0.001) and milk fat and protein concentrations were increased (P < 0.01) with ODM compared with TDM. Milk yield and fat and protein concentrations of milk from TDM and 13TWM herds were similar. Casein concentrations in ODM and TDM milks were similar, but ODM milk had a higher (P < 0.05) whey protein content. Somatic cell count of ODM and TDM milks was similar. Rennet coagulation time (RCT) and curd firmness (A60) of milk were not affected by milking frequency. However, rate of curd aggregation (K20) of ODM milk was reduced (P < 0.05) compared with that of TDM milk. Plasmin activity in ODM milk was numerically higher than in TDM milk, but the effect was not significant. ODM milk had higher NAGase activity than TDM milk (P < 0.01). In conclusion, once daily milking reduced milk yield by 29% and did not adversely affect the processability of milk. Moreover, one evening milking per week could be eliminated without adverse effects on milk yield or composition.