Effects of atropine on blood plasma amino acid profile and on the yields and concentration of milk components were investigated in 12 Friesian cows in early lactation. Cows were housed indoors and fed with cut pasture ad libitum. Each cow received four treatments over 12 d during a replicated 4×4 Latin square experiment. Treatments were: control (saline); low dose (L; 30 μg atropine/kg body weight (BW)); medium dose (M; 40 μg atropine/kg BW); and 2×L dose, 2 h apart (2×L). On each of four treatment days, cows were milked at about 7.00, after which treatments were administered by subcutaneous injection. Cows were milked again at 2 h, 6 h and 10 h after injection. Milk samples were collected at each milking. Immediately after the 2 h milking, blood samples were drawn from each cow and the second injection was given for the 2×L treatment. Atropine reduced hourly milk yield, and concentrations and hourly yields of total protein, casein, whey protein, α-casein, β-casein, κ-casein, β-lactoglobulin and α-lactalbumin, but by differing amounts. Milk concentrations of bovine serum albumin and immunoglobulin G were increased by atropine, and overall yields of these proteins were mostly unchanged. Atropine lowered concentrations of most, but not all, amino acids in blood plasma, with essential amino acids reduced more than non-essential amino acids. Concentrations of α-amino N in whole blood, and glucose and insulin in blood plasma, fell after atropine injection. There was no difference between the L and M doses of atropine, but the 2×L dose had greater effects on milk composition than the single doses. For yields of milk and milk components, the effect of the 2×L dose was also more persistent. The results highlight the differential synthesis of individual milk proteins, and suggest that atropine might be useful for evaluating the mechanisms regulating milk protein composition.