Purpose The aim of this study was to longitudinally compare novice nurses’ sleep disturbance, sleep hygiene, and physical activity and analyze factors affecting sleep disturbance. Methods Data were obtained from three follow-up surveys of 178 novice nurses. The data were analyzed using SPSS 25.0 to calculate descriptive statistics. A repeated measures ANOVA and Cochran’s Q test were performed, and a logistic regression analysis was conducted using generalized estimation equations. Results The sleep disturbance rate increased significantly over time in the following order: before the start of shift work, after six months of shift work, and after 18 months of shift work (T=98.67, p<.001). Sleep hygiene significantly deteriorated over time after the start of shift work (F=127.95, p<.001). Arousal behavior (OR=10.34, p<.001) and the sleep environment (OR=1.54, p=.005) in sleep hygiene, significantly influenced sleep disturbance. Stretching exercises were an influencing factor in sleep disturbance (OR=1.71, p=.039). Conclusion Sleep disturbance among novice nurses increased after the start of shift work. Sleep hygiene and stretching were influencing factors. Therefore, to prevent and reduce sleep disturbance in novice nurses, efforts are needed to improve sleep hygiene and promote stretching exercises.