Our study objective was to determine the effect of a short feed restriction (4 d) and subsequent refeeding (4 d) on the performance and metabolism of beef cows with a different nutritional status by particularly focusing on their milk fatty acid (FA) profile, to consider its potential use as biomarker of metabolic status. Thirty-two Parda de Montaña multiparous lactating beef cows were individually fed a diet based on the average cow’s net energy (NE) and metabolisable protein requirements. At 58 days in milk (DIM, d 0), cows underwent a 4 d feed restriction (55% requirements, restriction period). Before and after the restriction, diets met 100% of their requirements (basal and refeeding periods). Cow performance, milk yield and composition, and plasma metabolites, were determined on d -2, 1, 3, 5, 6, and 8. Cows were classified into two status clusters according to their pre-challenge performance and energy balance (EB) (Balanced vs. Imbalanced). All traits were statistically analyzed considering the fixed effect of status cluster and feeding period or day, with cow as a random effect. Imbalanced cows were heavier and had a more negative EB (P < 0.001), but similar milk yield, milk composition, and circulating metabolites (except for greater urea) than Balanced cows (P > 0.10). Milk contents of C18:1 cis-9, monounsaturated FA (MUFA), and mobilization FA were greater (P < 0.05), whereas saturated FA (SFA) and de novo FA were lesser in Imbalanced than Balanced cows (P < 0.05). Restriction decreased body weight (BW), milk yield, and milk protein compared to the basal period, but increased milk urea and plasma nonesterified fatty acids (NEFA) (P < 0.001). Milk contents of SFA, de novo, and mixed FA decreased immediately during the restriction, while MUFA, polyunsaturated FA and mobilization FA increased (P < 0.001). Basal milk FA contents were recovered on d 2 of refeeding, and all their changes strongly correlated with differences in EB and NEFA (P < 0.05). The general lack of interactions between status clusters and feeding periods implied that the response mechanisms to diet changes did not differ between cows with a different pre-challenge nutritional status.