False negative tests for SARS-CoV-2 are common and have important public health and medical implications. We tested the hypothesis of diurnal variation in viral shedding by assessing the proportion of positive versus negative SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) tests and cycle time (Ct) values among positive samples by the time of day. Among 86,342 clinical tests performed among symptomatic and asymptomatic patients in a regional health care network in the southeastern United States from March to August 2020, we found evidence for diurnal variation in the proportion of positive SARS-CoV-2 tests, with a peak around 1400 h and 1.7-fold variation over the day after adjustment for age, sex, race, testing location, month, and day of week and lower Ct values during the day for positive samples. These findings have important implications for public health testing and vaccination strategies.