ABSTRACT: We have previously showed that whey protein hydrolysate (WPH) causes a greater increase in muscle protein synthesis than an identical composition of amino acids mixture does. The present study was conducted to investigate a comparative effect of WPH on gene expression. Male Sprague-Dawley rats subjected to a 2-h swimming exercise were administered either a carbohydrate-amino acid diet or a carbohydrate-WPH diet immediately after exercise. One hour after exercise, epitrochlearis muscle mRNA was sampled and subjected to DNA microarray analysis. As a result, ingestion of WPH altered 189 genes in considering the false discovery rate. Among the upregulated genes, 8 Gene Ontology (GO) terms were enriched, which included key elements in muscle repair after exercise such as Cd24, Ccl2, Ccl7 and Cxcl1. On the other hand, 9 GO terms were enriched in the gene sets downregulated by ingestion of WPH and these GO terms fell into 2 clusters, 'regulation of ATPase activity' and 'immune response'. Furthermore, we found that WPH activate the 2 upstream proteins, extracellular signal-regulated kinase 1/2 (ERK1/2) and hypoxia-inducible factor-1a (HIF-1a), which may act as key factors for regulation of gene expression. These results suggest that ingestion of WPH, compared to an identical composition of amino acid mixture, induces greater changes in the after-exercise gene expression profile via activation of the proteins, ERK1/2 and HIF-1a. Male Sprague-Dawley rats (5-week-old) with body weights of approximately 150 g each (CLEA Japan, Inc., Tokyo, Japan) were used in this study. Rats were maintained at 23 +/- 2C, with lights on from 8:00 to 20:00 and off from 20:00 to 8:00. Rats had free access to food (protein 23.6%, fat 5.3%, carbohydrate 54.4%, ash 6.1%, fiber 2.9%, moisture 7.7%, MF, Oriental Yeast Co., Ltd., Osaka, Japan) and water. After 2- or 3-day acclimation, the rats were pre-trained to swimming exercise for 3 days. One day before the experiment, they were fed 5 g of a restricted diet (MF, Oriental Yeast Co., Ltd., Osaka, Japan). On the day of the experiment, rats swam for 2 hours, with 4 rats swimming simultaneously in a barrel filled with water to a depth of 50 cm, allowing an average surface area of 400 cm2 for each animal. The water temperature was maintained at a constant of 35C. Immediately following exercise, rats were given one of two isoenergetic test solutions by gavage. These solutions contained 44 kJ in a four-test dose that represented about 15% of daily energy needs and included either a mixed meal containing carbohydrate and amino acid mixture (AAM) or a mixed meal containing carbohydrate and whey protein hydrolysate (WPH; Meiji Co., Ltd., Japan). This study was approved by the Animal Committee of Food Science Research Lab., Meiji Co., Ltd., with the animals receiving care according to the guidelines laid down by this committee.