Currently, different protocols are used in various countries to estimate the 24-h fat and protein percentages for dairy cows that are milked with an automatic milking system. These protocols include estimating 24-h fat and protein percentages from 1) single samples from all animals in the herd; 2) single samples of fat adjusted for covariates and single samples of protein unadjusted for covariates; or 3) all samples (adjusted and unadjusted) collected on test days where test days vary in length from 10 to 18 h. The accuracy of estimated component percentages in predicting observed percentages was determined via agreement analysis for four protocols. Analysis of the data suggests collecting all samples (unadjusted for covariates) during a sampling period of at least 16 h on test day to be the most accurate protocol when estimating 24-h fat and protein percentages in herds with automatic milking systems.