The aim of the present study was to explore the relation of controlled dietary acrylamide (AA) intake with the excretion of AA-related urinary mercapturic acids (MA), N-acetyl-S-(carbamoylethyl)-l-cysteine (AAMA) and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-l-cysteine (GAMA). Excretion kinetics of these short-term exposure biomarkers were monitored under strictly controlled conditions within a duplicate diet human intervention study. One study arm (group A, n = 6) ingested AA via coffee (0.15–0.17 µg/kg bw) on day 6 and in a meal containing an upper exposure level of AA (14.1–15.9 μg/kg bw) on day 10. The other arm (group B) was on AA minimized diet (washout, 0.05–0.06 µg/kg bw) throughout the whole 13-day study period. On day 6, these volunteers ingested 13C3D3-AA (1 μg/kg bw). In both arms, urinary MA excretion was continuously monitored and blood samples were taken to determine hemoglobin adducts. Ingestion of four cups of coffee resulted in a slightly enhanced short-term biomarker response within the background range of group B. At the end of the 13-day washout period, group B excreted an AAMA baseline level of 0.14 ± 0.10 µmol/d although AA intake was only about 0.06 µmol/d. This sustained over-proportional AAMA background suggested an endogenous AA baseline exposure level of 0.3–0.4 µg/kg bw/d. The excretion of 13C3D3-AA was practically complete within 72–96 h which rules out delayed release of AA (or any other MA precursor) from deep body compartments. The results provide compelling support for the hypothesis of a sustained endogenous AA formation in the human body.