Objective/Background: Exercise training has been demonstrated to beneficially influence mean-level measures of sleep; however, few studies have examined the impact of an exercise intervention on night-to-night variability in sleep. This study investigated whether four months of moderate-intensity exercise impacted night-to-night variability in sleep among older women. Methods: Participants (n = 49) were randomized to one of two moderate-intensity walking programs with different doses of energy expenditure: low-dose (n = 23: 8 kcal/kg of body weight per week) or high-dose (n = 26: 14 kcal/kg of body weight per week). Sleep parameters were assessed objectively via actigraphy at baseline, mid- (2 months), and postintervention (4 months). Nightly variability in each of the sleep parameters was calculated using the seven-day standard deviation (SD) and a coefficient of variation (SD/mean x 100%). Cardiorespiratory fitness (VO2peak) was measured at baseline and postintervention using a graded treadmill test. Results: Both measures of nightly variability demonstrated a borderline to significantly lower amount of night-to-night variability in wake time after sleep onset (WASO) and number of awakenings at postintervention in comparison to baseline (p ≤ 0.05). Higher VO2peak levels at baseline were associated with less time in bed and lower total sleep time variability throughout the exercise intervention (p < 0.05). Conclusion: Overall, participation in moderate-intensity exercise was observed to reduce the amount of nightly variability for WASO and number of awakenings over time in older women.