Temporal pattern of differential gene expression is associated with changes in milk yield. Cows were assigned to unilateral frequent milking (UFM; twice daily milking (2×) of the left udder half, four times daily milking (4×) of the right udder half) during d 1 to 21 of lactation. Mammary biopsies were obtained from both udder halves on d 21, 23, and 40 of lactation. Gene expression was detected using Affymetrix GeneChip® Bovine Genome Arrays. Genes of which differential expression changed over time were grouped into 4 clusters based on the temporal pattern of differential expression. Cluster 1 (panel A; 61genes) included genes whose expression in 4× vs 2× udder halves was lower on day 21, higher on day 23 (after cessation of 4× milking), and subsequently lower on day 40 of lactation. Cluster 2 (panel B; 3 genes) included genes whose expression in 4× vs. 2× udder halves was lower on day 21, higher on day 23, and remained higher on day 40 of lactation. Cluster 3 (panel C; 8 genes) included genes whose expression in 4× vs. 2× udder halves was higher on day 21, lower on day 23, and higher on day 40. Finally, cluster 4 (panel D; 3 genes) included genes whose expression in 4× vs. 2× udder halves was higher on day 21, lower on day 23, and remained lower on day 40. Differential milk yield is plotted on the left Y-axis and differential expression of genes in each cluster is plotted on the right Y-axis. Gene expression data are presented as signed fold change (4× minus 2×) for each gene. Solid vertical line represents cessation of UFM and return of all 4 glands to 2× milking on day 21 of lactation.