The aim of the study was to assess the effects of 5 supplements during a short-term period of negative energy and protein balance induced by feed restriction on expression of genes in liver and muscle. Seven multiparous Holstein cows (93 ± 15 DIM) were randomly assigned to 7 treatments in a 7 × 4 incomplete Latin square design with 5-d periods. Daily DMI was restricted to provide 60% of net energy requirements. Treatments were 4-h abomasal infusions of amino acid (AA) or glucose (0.4 mol/kg BW) initiated at feeding time (0900 h): monosodium glutamate (MSG), lysine (LYS), glutamine (GLN), valine (VAL), and water (CON) as control. Liver and muscle tissue was biopsied on the last day of the period. Total RNA was sequenced on an Ilumina HiSeq2500. Transcripts were assembled from the reads aligned to NCBI Bos taurus UMD_3.1.1. Statistical analysis was done using the “voom” method from the limma package in R. Data of differentially expressed genes between each treatment and CON with their associated P values were imported into DAVID and DIA for enrichment (↑ up- and ↓ downregulated) and functional analysis. Infusion of GLN overexpressed pathways for metabolism of AA (↑), gluconeogenesis (↑) [SDS], synthesis of pantothenate and CoA (↑), circadian rhythm regulation (↑) [PER1, PER2], immune system (↑) [S100A9, CXCR2]. Synthesis of serotonin and melatonin (↓) [TPH1], Tyr metabolism (↑), cholesterol transport (↑) [LDLR] and glucose metabolism (↑) [HK2, GFOD1] were the most significant pathways during MSG. Infusion of LYS affected TOR signaling (↑) [PRKG1, GPAT3], cholesterol synthesis (↓) [DHCR24], FA transport (↓) [FABP7], and immune system (↓) [GPR39, PADI2, C3]. Treatment of VAL upregulated mineral tissue turnover [ALPL, MN1] and possibly reduced muscle tissue mobilization [SESN2 (↑), CHSY3 (↓), ADAMTS5 (↓), MEOX2 (↓), LAMA2 (↓), CCNG1 (↑), DPP4 (↓), GTSE1 (↓)]. No relevant functional pathway was found during GLC. Our results identify some of the possible mechanisms of action that supplementation of these AA provided to counteract a period of negative energy and protein balance.