A single application of a high sun protection factor (SPF) sunscreen has been shown to prevent sunburn, but the existence of a further protective effect following re-application of sunscreen has received little attention. The aim of this study was to quantify the ultraviolet radiation (UVR) protection against sunburn afforded by a re-application of sunscreen relative to a single application. The study methods were based on the Australian Standard SPF testing regimen, using human volunteers, standardized applications of sunscreen and an artificial solar simulating UVR source. Sunscreen was initially applied, followed by a suberythemal UVR dose; sunscreen was then re-applied, followed immediately by a second period of controlled UV exposure sufficient to induce mild erythema. Compared to the first application, the second sunscreen application afforded 3.1 times more protection against minimal UVR-induced erythema. The combined effect of two sunscreen applications gave on average 2.3 times better protection from UVR than a single sunscreen application. Health authorities worldwide have recommended sunscreen re-application for the prevention of sunburn for some time. This is the first quantitative study to substantiate such recommendations.