Purpose: To determine whether the 2003 Accreditation Council for Graduate Medical Education (ACGME) duty hours reform affected medical knowledge as reflected by written board scores for internal medicine (IM) residents. Method: The authors conducted a retrospective cohort analysis of postgraduate year 1 (PGY-1) Internal Medicine residents who started training before and after the 2003 duty hour reform using a merged data set of American Board of Internal Medicine (ABIM) Board examination and the National Board of Medical Examiners (NMBE) United States Medical Licensing Examination (USMLE) Step 2 Clinical Knowledge test scores. Specifically, using four regression models, the authors compared IM residents beginning PGY-1 training in 2000 and completing training unexposed to the 2003 duty hours reform (PGY-1 2000 cohort, n = 5,475) to PGY-1 cohorts starting in 2001 through 2005 (n = 28,008), all with some exposure to the reform. Results: The mean ABIM board score for the unexposed PGY-1 2000 cohort (n = 5,475) was 491, SD = 85. Adjusting for demographics, program, and USMLE Step 2 exam score, the mean differences (95% CI) in ABIM board scores between the PGY-1 2001, 2002, 2003, 2004 and 2005 cohorts minus the PGY-1 2000 cohort were-5.43 (-7.63,-3.23),-3.44 (-5.65,-1.24), 2.58 (0.36, 4.79), 11.10 (8.88, 13.33) and 11.28 (8.98, 13.58) points respectively. None of these differences exceeded one-fifth of an SD in ABIM board scores. Conclusions: The duty hours reforms of 2003 did not meaningfully affect medical knowledge as measured by scores on the ABIM board examinations.