More than 2 million people in the United States have myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We performed targeted, broad-spectrum metabolomics to gain insights into the biology of CFS. We studied a total of 84 subjects using these methods. Forty-five subjects (n = 22 men and 23 women) met diagnostic criteria for ME/CFS by Institute of Medicine, Canadian, and Fukuda criteria. Thirty-nine subjects (n = 18 men and 21 women) were age- and sex-matched normal controls. Males with CFS were 53 (±2.8) y old (mean ± SEM; range, 21-67 y). Females were 52 (±2.5) y old (range, 20-67 y). The Karnofsky performance scores were 62 (±3.2) for males and 54 (±3.3) for females. We targeted 612 metabolites in plasma from 63 biochemical pathways by hydrophilic interaction liquid chromatography, electrospray ionization, and tandem mass spectrometry in a single-injection method. Patients with CFS showed abnormalities in 20 metabolic pathways. Eighty percent of the diagnostic metabolites were decreased, consistent with a hypometabolic syndrome. Pathway abnormalities included sphingolipid, phospholipid, purine, cholesterol, microbiome, pyrroline-5-carboxylate, riboflavin, branch chain amino acid, peroxisomal, and mitochondrial metabolism. Area under the receiver operator characteristic curve analysis showed diagnostic accuracies of 94% [95% confidence interval (CI), 84-100%] in males using eight metabolites and 96% (95% CI, 86-100%) in females using 13 metabolites. Our data show that despite the heterogeneity of factors leading to CFS, the cellular metabolic response in patients was homogeneous, statistically robust, and chemically similar to the evolutionarily conserved persistence response to environmental stress known as dauer.