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