Plant factories with artificial lighting have been developed to improve food production, functional ingredients, and profitability. Intensive research has been performed to elucidate the effects of light intensity and wavelength on plant growth and nutritional quality with the use of light-emitting diodes (LEDs). In particular, the effects of monochromatic red, blue, or simultaneous red + blue light have been studied because these wavelengths are predominantly used for photosynthesis. We examined the effects of alternating red and blue light provided by LEDs over a period of 24 hours on the growth and nutritional properties of leafy lettuce. The results clearly show that alternating red and blue light accelerated plant growth significantly compared with white fluorescent lamps or red and blue LEDs at the same daily light integral. Plants grown under alternating red/blue light had a greater net assimilation rate and total and projected leaf area (an indicator of the fraction of leaf area that absorbs more light) than other plants. Additionally, alternating red and blue light maintained high concentrations of sugars, ascorbic acid, and anthocyanins in leaves. Taken together, the results indicate that continuous irradiation with alternating red and blue light could enhance growth while maintaining the nutritional quality in lettuce.