This work was designed to investigate whether the profile of non-esterified fatty acids (NEFA) remains constant in response to underfeeding in beef cows, since in other species mobilization of fatty acids (FA) has been shown to be dependent on fatty acid structure. Twenty-one Charolais cows (multiparous, no. = 16 and primiparous, no. = 5) were assigned to one of two diets (control diet (CD; no. = 11) or restricted diet (RD; no. = 10) which, respectively supplied all or 0·7 of energy and protein requirements) after calving. Blood and composite milk samples were collected weekly before the morning meal over a period of 9 weeks. The cows given RD lost significantly more body weight (P < 0·001) and condition score (P < 0·05) than the cows given CD. Plasma NEFA and urea concentrations were also higher in RD than CD during the experiment (P < 0·05 and P < 0·001, respectively). The level of feeding did not affect plasma insulin and glucose concentrations. Milk FA were affected by feeding level. The proportions of short- and medium-chain FA were lower in RD compared with CD (C8: 0, C10: 0, C12: 0 and C14: 0, P < 0·05 and C14: 1 and C16: 0, P < 0·10) while some of the long-chain FA were higher (C17: 0 and C18: 1, P < 0·001 and C17: 1, P < 0·10). Feeding level affected the profile of FA in plasma NEFA. The proportions of C16: 0 and C18: 1 were higher (P < 0·05) in RD cows compared with CD cows while the proportions of C12: 0, C14: 1, C17: 0, C18: 0 and C18: 2 were lower (P < 0·05). There was a feeding level × time interaction in the proportions of different FA in the plasma NEFA and in the total plasma NEFA concentrations. The differences caused by feeding level gradually disappeared with time. The proportion of C18: 1 in plasma NEFA and the total plasma NEFA concentrations were quadratically positively correlated, while the correlation between C18: 2 and total plasma NEFA was negative. In conclusion, when post-partum beef cattle are underfed, plasma NEFA concentrations increase (indicating lipomobilization) and this increase is associated with an increase in the proportion of oleic acid (C18: 1) in plasma NEFA.