Excited fluorophores produce reactive oxygen species that are toxic toward many live cells (phototoxicity) and accelerate bleaching of the fluorophores during the course of extended or repeated measurements (photobleaching). We recently developed an illumination system for fluorescence microscopy using a high power light-emitting diode (LED), which can emit short pulses of light (0.5-2 ms) to excite fluorophores. This system minimizes illumination time, thus reducing phototoxicity and photobleaching artifacts. To demonstrate the usefulness of the new system, we compared images of human sperm loaded with various fluorescent indicators and excited with either a conventional mercury lamp as a continuous excitation light source or the LED as a source of pulsed illumination. We found that sperm motility decreased rapidly and photobleaching was relatively rapid under continuous illumination, whereas under pulsed LED illumination, motility was maintained and photobleaching was much reduced. Therefore, fluorescence microscopy using LED-based pulsed illumination offers significant advantages for long-term live cell imaging, reducing the degree of phototoxicity, and extending the effective lifetime of fluorophores.