BACKGROUND: High ambient temperatures are associated with an acute increase in mortality risk. Although heat exposure during the night is anecdotally cited as being important, this has not been rigorously demonstrated in the epidemiological literature. METHODS: We quantified the contribution of nighttime temperatures using time-series quasi-Poisson regression on cause and age-specific daily mortality in London between 1993 and 2015. Daytime and nighttime exposures were characterized by average temperatures between 9 am and 9 pm and between 4 am and 8 am, respectively, lagged by 7 days. We also examined the differential impacts of hot and cool nights preceded by very hot days. All models were adjusted for air quality, season, and day of the week. Nighttime models were additionally adjusted for daytime exposure. RESULTS: Effects from nighttime exposure persisted after adjusting for daytime exposure. This was highest for stroke, RR (relative risk) = 1.65 (95% confidence interval (CI) = 1.27 to 2.14) estimated by comparing mortality risk at the 80th and 99th temperature percentiles. Compared to daytime exposure, nighttime exposure had a higher mortality risk on chronic ischemic and stroke and in the younger age groups. Respiratory mortality was most sensitive to daytime temperatures. Hot days followed by hot nights had a greater mortality risk than hot days followed by cool nights. CONCLUSIONS: Nighttime exposures make an additional important contribution to heat-related mortality. This impact was highest on warm nights that were preceded by a hot day, which justifies the alert criteria in heat-health warning system that is based on hot days followed by hot nights. The highest mortality risk was from stroke; targeted interventions would benefit patients most susceptible to stroke.