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There is evidence that the spring Daylight Saving Time (DST) transition acutely increases motor vehicle accident (MVA) risk (“DST effect”), which has been partly attributed to sleep deprivation and circadian misalignment []. Because spring DST also shifts clock time 1 h later, mornings are darker and evenings brighter, changing illumination conditions for peak traffic density. This daytime-dependent illumination change (“time of day effect”) is hypothesized to result in DST-associated afternoon and evening accident risk reductions []. Furthermore, sunrise and local photoperiod timing depend on position in time zone. The sun rises at an earlier clock time in the eastern regions of a given time zone than in the western regions, which is thought to induce higher levels of circadian misalignment in the west than in the east (“time zone effect”) []. This study evaluated the acute consequences of the DST transition on MVAs in a chronobiological context, quantifying DST, time of day, and time zone effects. We used large US registry data, including 732,835 fatal MVAs recorded across all states (1996–2017), and observed that spring DST significantly increased fatal MVA risk by 6%, which was more pronounced in the morning and in locations further west within a time zone. DST-associated MVA risk increased even in the afternoon hours, despite longer daylight hours. The MVA risk increase waned in the week subsequent to DST, and there were no effects of the fall-back transition to Standard Time (ST) on MVA risk, further supporting the hypothesis that DST-transition-associated, preventable circadian misalignment and sleep deprivation might underlie MVA risk increases.