Our aim was to evaluate the effects of once-daily milking on the welfare of dairy cows in a pastoral-based farming system. There are concerns that cows milked only once daily may experience discomfort associated with udder distension. We evaluated the behavior, including lying time and postures, grazing patterns, and kicking behavior in the parlor, and measures of udder firmness in cows milked once daily in 2 experiments: 1) at peak lactation, comparing cows milked once (1 x) or twice-daily (2 x) from the time of calving (n = 4 groups; 1 x and 2 x treatments); and 2) during the transition from 2 x to 1 x milking at mid lactation (n = 12 groups; 1 x, 2 x, and 2 x to 1 x treatments or TRANS). At peak lactation, cows milked 1 x were more likely to lie with their hind legs touching the body than cows milked 2 x. There were no differences in udder firmness (1 x: 10.0 vs. 2 x: 9.9 +/- 0.13 g of force) or kicking (1 x: 25 vs. 2 x: 24 +/- 7.8%) behavior in the parlor. In the second experiment, milking frequency was reduced from 2 x to 1 x and this change resulted in an increase in udder firmness (1 x: 9.1, 2 x: 9.3, and TRANS: 9.5 +/- 0.14 g of force). Despite these differences in udder firmness, lying time (1 x: 10.1, 2 x: 9.5, and TRANS: 9.6 +/- 0.38 h/24 h), postures, and concentrations of fecal glucocorticoid metabolites (1 x: 10.0, 2 x: 10.5, and TRANS: 10.4 +/- 0.49 ng/g of feces in the 3 d after transition) in cows milked 1 x or 2 x were similar. Although milking frequency did not influence total time spent grazing, the diurnal grazing pattern of cows milked 2 x was disrupted by afternoon milking at both peak and mid lactation. Cows milked 2 x were able to compensate for this by grazing more after the return to pasture (DIM 153 to 155, 1 x: 52 vs. 2 x: 66 +/- 6.2 min/90 min of grazing). Cows milked 1 x had slightly longer strides than 2 x cows at mid lactation (1 x: 0.73, 2 x: 0.70, and TRANS: 0.70 +/- 0.014 strides/m). In conclusion, under our experimental conditions, found no overt changes in behavior or physiology to indicate that the welfare of cows milked 1 x is impaired.