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Background: In animals, dietary sulfur amino acid restriction (SAAR) improves metabolic health, possibly mediated by altering sulfur amino acid metabolism and enhanced anti-obesogenic processes in adipose tissue. Aim: To assess the effects of SAAR over time on the plasma and urine SAA-related metabolites (sulfurome) in humans with overweight and obesity, and explore whether such changes were associated with body weight, body fat and adipose tissue gene expression. Methods: Fifty-nine subjects were randomly allocated to SAAR (∼2 g SAA, n = 31) or a control diet (∼5.6 g SAA, n = 28) consisting of plant-based whole-foods and supplemented with capsules to titrate contents of SAA. Sulfurome metabolites in plasma and urine at baseline, 4 and 8 weeks were measured using HPLC and LC-MS/MS. mRNA-sequencing of subcutaneous white adipose tissue (scWAT) was performed to assess changes in gene expression. Data were analyzed with mixed model regression. Principal component analyses (PCA) were performed on the sulfurome data to identify potential signatures characterizing the response to SAAR. Results: SAAR led to marked decrease of the main urinary excretion product sulfate (p