Introduction: Chest pain is one of the most common presenting complaints to emergency departments (EDs) across the world, and the exclusion of acute myocardial infarction (AMI) using troponin testing is central to the care of many of these patients. While testing strategies using conventional troponin assays require repeat testing over many hours to avoid missed diagnoses, high-sensitivity troponin (hs-cTn) assays may be able to exclude AMI in a large proportion of patients with a single result at presentation. This objective of this study is to validate the ability of very low concentrations of hs-cTn at presentation to exclude AMI in ED chest pain patients. Methods: This prospective cohort study was conducted at a single urban tertiary center and regional percutaneous coronary intervention site in Calgary, Alberta. Patients were eligible for enrolment if they presented to the ED with chest pain, were 25-years or older and required biomarker testing to rule out AMI at the discretion of the attending emergency physician. Patients were excluded if they had clear acute ischemic ECG changes, new arrhythmia or renal failure requiring hemodialysis. A high-sensitivity troponin result (Roche Elecsys hs-cTnT) was obtained in all patients at presentation. The primary outcome was AMI on the index visit. Secondary outcomes included 30-day AMI and major adverse cardiac events (MACE - including AMI, revascularization or cardiac death). Electronic medical records were reviewed and telephone follow-up was obtained for all patients at 30-days to ensure relevant events were captured. Two physician adjudication (board-certified emergency physician and cardiologist) was obtained for all outcomes. This study was REB approved. Results: A total of 1,167 patients were enrolled from August 2014 September 2016, of which 191 (16.3%) patients had an initial troponin below the limit of blank (LoB, <3 ng/L) and 416 (32.8%) were below the limit of detection (LoD, <5 ng/L). The sensitivity of a single troponin below the LoB (<3 ng/L) for index AMI was 100% (95% CI 96.2%-100%) and for 30-day AMI was 100% (95% CI 96.4-100%). The sensitivity of a troponin below the LoD (<5 ng/L) for index AMI was 97.9% (95% CI 92.7%-99.8%) and for 30-day AMI was 98.0% (95% CI 93.0-99.8%). Sensitivity for 30-day MACE at both cutoffs was lower: 98.4% (95% CI 94.3-99.8%) for <3 ng/L, and 94.4% (95% CI 88.8-97.7%) for <5 ng/L, respectively; however, negative predictive values remained high at both cutoffs: <3 ng/L, 99.0% (95% CI 96.3-99.9%) and <5 ng/L, 98.3% (95% CI 96.6-99.3%). Conclusion: A high sensitivity troponin T result below the LoB (<3 ng/L) is highly sensitive for excluding AMI and identifies patients at low risk of 30-day MACE. A result below the LoB (<5 ng/L) will identify a larger population of patients as low risk but has a greater risk of missed AMI and MACE.