The aim of the study was to determine metabolic responses to a short-term period of negative energy balance induced by feed restriction (FR). 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 energy requirements; the 7th treatment consisted of ad libitum (AL) intake. Feed was provided once daily at 0900 h. Effects of FR (AL vs RC), day, time within day, and interactions were evaluated using the MIXED procedure of SAS. Milk yield (P < 0.01), milk protein concentration (P = 0.03) and yield (P < 0.01), and lactose yield (P < 0.01) were lower for RC, whereas milk fat (P < 0.01) and urea N concentrations were higher (P < 0.01). Treatment RC induced lower plasma insulin (P = 0.01) and glucose (P = 0.04) concentrations, with quadratic (P < 0.01 for both) decreasing trends reaching nadir on d 3. Concentration of NEFA was higher (P < 0.01) and increased quadratically (P < 0.01) with its maximum on d 3 during FR. Serum BHB increased linearly (P = 0.04) for RC (RC × d; P = 0.16) with its peak at d 4. Catabolism of amino acids (AA) increased early during FR as indicated by plasma urea N increasing (P < 0.01) quadratically (P < 0.01), with its peak on d 2 and decreasing afterward. Plasma 3-methylhistidine increased linearly (P < 0.01) denoting tissue mobilization. A group of AA (Glu, Val, Leu, Tyr, Phe, Ser, His, Thr, Asn, Ala, Pro, Met) decreased in a quadratic manner with the nadir at d 2 and 3, while Asp, Trp and Ile decreased linearly. Concentrations of other AA increased (Gln, Gly, Cys) or did not vary (Lys, Arg) during FR. Plasma AA concentrations decreased after feed delivery in both diets, coinciding with the increase of insulin, except for Glu and Gln that increased after feeding only during FR. Metabolic adaptations to lower insulin during FR seemed to select catabolism of AA as the first energy source before later relying more on fatty acids. Based on responses of plasma AA and insulin to feeding, protein synthesis in tissues likely remained sensitive to insulin within day.