The objective was to study the effects of week of lactation (WOL) and experimental nutrient restriction on concentrations of selected milk metabolites and fatty acids (FA), and assess their potential as biomarkers of energy status in early-lactation cows. To study WOL effects, 17 multiparous Holstein cows were phenotyped from calving until 7 WOL while allowed ad libitum intake of a lactation diet. Further, to study the effects of nutrient restriction, 8 of these cows received a diet containing 48% straw (high-straw) for 4 d starting at 24 ± 3 days in milk (mean ± SD), and 8 cows maintained on the lactation diet were sampled to serve as controls. Blood and milk samples were collected weekly for the WOL data set, and daily from d -1 to 3 of nutrient restriction (or control) for the nutritional challenge data set. Milk β-hydroxybutyrate (BHB), isocitrate, glucose, glucose-6-phosphate (glucose-6P), galactose, glutamate, creatinine, uric acid, and N-acetyl-β-D-glucosaminidase activity (NAGase) were analyzed in p.m. and a.m. samples, and milk FA were analyzed in pooled p.m. and a.m. samples. Average energy balance (EB) per day ranged from -27 MJ/d to neutral when cows received the lactation total mixed ration, and from -109 to -87 ± 7 MJ/d for high-straw (least squares means ± standard error of the mean). Plasma nonesterified FA concentration was 1.67 ± 0.13 mM and BHB was 2.96 ± 0.39 mM on the d 3 of high-straw (least squares means ± standard error of the mean). Milk concentrations of BHB, glucose, glucose-6P, glutamate, and uric acid differed significantly between p.m. and a.m. milkings. Milk isocitrate, glucose-6P, creatinine, and NAGase decreased, whereas milk glucose and galactose increased with WOL. Changes in milk BHB, isocitrate, glucose, glucose-6P, and creatinine were concordant during early lactation and in response to nutrient restriction. Milk galactose and NAGase were modulated by WOL only, whereas glutamate and uric acid concentrations responded to nutrient restriction only. The high-straw increased milk concentrations of FA potentially mobilized from adipose tissue (e.g., C18:0 and cis-9 C18:1 and sum of odd- and branched-chain FA (OBCFA) with carbon chain greater than 16; ∑ OBCFA >C16), and decreased concentrations of FA synthesized de novo by the mammary gland (e.g., sum of FA with 6 to 15 carbons; ∑ C6:0 to C15:0). Similar observations were made during early lactation. Plasma nonesterified FA concentrations had the best single linear regression with EB (R2 = 0.62). Milk isocitrate, Σ C6:0 to C15:0. and cis-9 C18:1 had the best single linear regressions with EB (R2 ≥ 0.44). Milk BHB, isocitrate, galactose, glutamate, and creatinine explained up to 64% of the EB variation observed in the current study using multiple linear regression. Milk concentrations of ∑ C6:0 to C15:0, C18:0, cis-9 C18:1, and ∑ OBCFA >C16 presented some of the best correlations and regressions with other indicators of metabolic status, lipomobilization, and EB, and their responses were concordant during early lactation and during experimental nutrient restriction. Metabolites and FA secreted in milk may serve as noninvasive indicators of metabolic status and EB of early-lactation cows.