Sampling for analysis of milk constituents (fat and protein) is a routine procedure on commercial dairy farms. It is assumed that for this procedure a representative sample of whole milk is taken, either as a fractionated sample from the milk meter or as a thoroughly mixed milk sample taken after the milk has been agitated with air in the recording jar. The use of on-line sensors for milk analysis will provide real-time data on which nutritional management decisions can be made according to milk composition. However, there is no recommendation as to the optimum sampling time in an on-line system to obtain a representative sample during the milking process. Ideally sampling should be carried out as early as possible for the analysis to be completed before the milking system is needed for the next cow. It is important to establish within milking variation of milk constituents for an on-line sensing system, especially since fat increases significantly throughout milking. The aims of this study were to determine the within milking variation of urea, acetone, fat and protein and to specify the most appropriate time of sampling within milking to obtain representative concentrations of these constituents and analytes.