The aim of the study was to assess the effects of a short-term period of negative energy balance (NEB) 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. In 6 treatments, including a restricted control (RC), daily DMI was restricted to provide 60% of calculated net energy requirements in the 7th treatment cows were fed for ad libitum (AL) DMI. 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 separately for each tissue using the “voom” method from the limma package in R. Data of differentially expressed genes (DEG) between RC and AL with their associated P values were imported into DAVID and DIA for enrichment (↑ up- and ↓ downregulated) and functional analysis. A total of 284 genes in liver (FDR P < 0.25) and 261 in muscle (uncorrected P < 0.01) were detected. The top most significant categories among the 3 gene ontology (GO), KEGG and Reactome pathways were related to regulation of metabolism and signaling [insulin, AMPK and FoxO signaling, cell death and apoptosis, PPAR (↑) and glucagon signaling, synaptic plasticity (↓)], lipid metabolism [cell lipid transporters, peroxisome, cholesterol transport, fat digestion and absorption (↑)], AA metabolism [biosynthesis of AA (Lys ↑), urea cycle (↑)], vitamin metabolism [vitamin absorption and digestion, folate biosynthesis (↓)], and carbohydrate metabolism (Liver: ↑; muscle ↓) [pentose, ascorbate and aldarate], immune system (↓), calcium homeostasis (↑), and endocrine metabolism (estrogen, thyroid and oxytocin signaling). The genes with the greatest log2 fold change emphasized the vital roles of lipid metabolism [APOA1, HMGCS2, BREH1, and FABP7 (↑)], immune system [SAA1 (↓)], protein and bone mobilization [SERPINA3 (↓), PI16 (↑), SPP2 (↑)], and vitamin B metabolism [TCN1 (↓)].