Importance: Glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1) have been widely studied and show promise for clinical usefulness in suspected traumatic brain injury (TBI) and concussion. Understanding their diagnostic accuracy over time will help translate them into clinical practice. Objectives: To evaluate the temporal profiles of GFAP and UCH-L1 in a large cohort of trauma patients seen at the emergency department and to assess their diagnostic accuracy over time, both individually and in combination, for detecting mild to moderate TBI (MMTBI), traumatic intracranial lesions on head computed tomography (CT), and neurosurgical intervention. Design, Setting, and Participants: This prospective cohort study enrolled adult trauma patients seen at a level I trauma center from March 1, 2010, to March 5, 2014. All patients underwent rigorous screening to determine whether they had experienced an MMTBI (blunt head trauma with loss of consciousness, amnesia, or disorientation and a Glasgow Coma Scale score of 9-15). Of 3025 trauma patients assessed, 1030 met eligibility criteria for enrollment, and 446 declined participation. Initial blood samples were obtained in 584 patients enrolled within 4 hours of injury. Repeated blood sampling was conducted at 4, 8, 12, 16, 20, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, and 180 hours after injury. Main Outcomes and Measures: Diagnosis of MMTBI, presence of traumatic intracranial lesions on head CT scan, and neurosurgical intervention. Results: A total of 1831 blood samples were drawn from 584 patients (mean [SD] age, 40  years; 62.0%[362 of 584] male) over 7 days. Both GFAP and UCH-L1 were detectible within 1 hour of injury. GFAP peaked at 20 hours after injury and slowly declined over 72 hours. UCH-L1 rose rapidly and peaked at 8 hours after injury and declined rapidly over 48 hours. Over the course of 1 week, GFAP demonstrated a diagnostic range of areas under the curve for detecting MMTBI of 0.73 (95%CI, 0.69-0.77) to 0.94 (95%CI, 0.78-1.00), and UCH-L1 demonstrated a diagnostic range of 0.30 (95%CI, 0.02-0.50) to 0.67 (95%CI, 0.53-0.81). For detecting intracranial lesions on CT, the diagnostic ranges of areas under the curve were 0.80 (95%CI, 0.67-0.92) to 0.97 (95%CI, 0.93-1.00)for GFAP and 0.31 (95%CI, 0-0.63) to 0.77 (95%CI, 0.68-0.85) for UCH-L1. For distinguishing patients with and without a neurosurgical intervention, the range for GFAP was 0.91 (95%CI, 0.79-1.00) to 1.00 (95% CI, 1.00-1.00), and the range for UCH-L1 was 0.50 (95%CI, 0-1.00) to 0.92 (95%CI, 0.83-1.00). Conclusions and Relevance: GFAP performed consistently in detecting MMTBI, CT lesions, and neurosurgical intervention across 7 days. UCH-L1 performed best in the early postinjury period.