The aim of this study was to evaluate whether NMR-based metabolomics is a suitable method to generate an integrated view on metabolic processes during food deprivation in salmonid fish. Juvenile rainbow trout (Oncorhynchus mykiss) weighing 43-115 g were either fed ad libitum or deprived of food for 28 days at 10°C to investigate catabolic effects on energy reserves and metabolite patterns. The NMR analysis of plasma, liver, and muscle extracts revealed significant fasting-induced changes in the metabolome. Altered plasma lipoprotein levels and tissue-specific patterns of fatty acid mobilization were the most prominent responses, emphasizing the role of lipids as the primary energy source during fasting. In plasma, very-low-density lipoprotein levels increased in food-deprived fish compared with fed fish, whereas levels of high-density lipoprotein decreased. Signs of muscle protein catabolism were also observed as changes in the alanine dynamics. The results further suggest that mechanisms to preserve liver glycogen are present in the food-deprived fish. This study thus demonstrates the utility of NMR-based metabolomics of tissue extracts and plasma to describe the integrated metabolic status of fish.