The objective of this study was to measure the effects of varying doses of atropine on the concentration and composition of milk protein and on blood α-amino N levels. Four treatments were administered to each of 12 cows over 12 days in a replicated Latin square experiment. There were at least 2 days between each of 4 treatment days. Treatments were: control (C; saline); low dose (L; 30mg atropine/kg LWT); medium dose (M; 40mg atropine/kg LWT); and 2 x low dose (2L). All treatments were administered via subcutaneous injection immediately after the morning milking; the second dose of the 2L treatment was given two hours later. Milk was sampled from each cow at the morning milking (time 0 h). Cows were then milked again 2, 6 and 10 h after treatment, and milk samples again collected. Blood samples were drawn from the coccygeal vein of each cow after each milking. Atropine decreased milk secretion at 6 h for the 2L treatment and 10 h for all treatments. Atropine reduced concentrations of milk protein and casein at 2 h and 6 h, but not at 10 h. Concentrations of whey proteins and of α-casein were depressed by atropine only at 6 h post-treatment, while a reduction in α-lactalbumin due to atropine was observed at 6 and 10 h post-treatment. In contrast, atropine increased concentrations of IgG and BSA at 6 h and 10 h post-treatment. Atropine also increased the ratio of casein:total protein at 6 h after injection. There was no difference between the effects of the low and medium doses of atropine, but the double low dose induced effects which were greater than for the single doses. Effects of a single dose of atropine were greatest for most milk proteins at 6 h post treatment; thus this would be the most useful milk sampling time for future experiments. Atropine did not significantly affect α-amino N concentrations in whole blood, although there was a trend for a reduction for all treatments at 2 h after treatment. Atropine may be useful for reducing milk protein concentrations and circulating levels of certain blood amino acids to base levels, during studies designed to elucidate the effects of perturbations in the blood amino acid profile on milk protein composition.