Study Objectives: Nocturnal awakenings may constitute a unique risk for falls among older adults. We describe differences in gait and balance between presleep and midsleep testing, and whether changes in the lighting environment during the midsleep testing further affect gait and balance. Methods: Twenty-one healthy, late middle-aged and older (64.7 ± 8.0 y) adults participated in this repeated-measures design consisting of four overnight laboratory stays. Each night, participants completed baseline visual acuity, gait, and balance testing. After a 2-h sleep opportunity, they were awakened for 13 min into one of four lighting conditions: very dim white light (< 0.5 lux); dim white light (∼28.0 lux); dim orange light (∼28.0 lux); and white room-level light (∼200 lux). During this awakening, participants completed the same sequence of testing as at baseline. Results: Low-contrast visual acuity significantly decreased with decreasing illuminance conditions (F(3,45) = 98.26, p < 0.001). Our a priori hypothesis was confirmed in that variation in stride velocity and center of pressure path length were significantly worse during the mid-sleep awakening compared to presleep baseline. Lighting conditions during the awakening, however, did not influence these parameters. In exploratory analyses, we found that over one-third of the tested gait and balance parameters were significantly worse at the midsleep awakening as compared to baseline (p < 0.05), and nearly one-quarter had medium to large effect sizes (Cohen d ≥ 0.5; r ≥ 0.3). Conclusions: Balance and gait are impaired during midsleep awakenings among healthy, late middle-aged and older adults. This impairment is not ameliorated by exposure to room lighting, when compared to dim lights.