Circadian rhythms influence and coordinate an organism’s response to its environment and to invading pathogens. We studied the diurnal variation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasal/throat swabs collected in late 2020 to spring 2021 in a population immunologically naïve to SARS-CoV-2 and prior to widespread vaccination. SARS-CoV-2 diagnostic PCR data from 1698 participants showed a significantly higher viral load in samples obtained in the afternoon, in males, and in hospitalised patients when linear mixed modelling was applied. This study illustrates the importance of recording sample collection times when measuring viral replication parameters in clinical and research studies.