OBJECTIVE To determine the effect of overnight wear of contact lenses (CL) on tear meniscus, tear film stability, and subjective comfort. METHODS Sixty asymptomatic subjects were divided into three groups. The control group was composed of 20 non-CL wearers. Twenty inexperienced and 20 experienced CL wearers were enrolled as group 1 and group 2. For each subject in groups 1 and 2, a hydrogel and a silicone hydrogel CL were randomly assigned to each eye. Optical coherence tomography was used to determine volumes of the upper and lower tear menisci. Tear film stability was determined by non-invasive tear break-up time (NITBUT) measured by tearscope. A previously used questionnaire assessed dry eye symptoms. Subjects were evaluated during an 11-h period, including immediately before and after sleeping. RESULTS Compared with the presleep levels, tear meniscus volume in the control group increased immediately after awakening and recovered by 1 hour later (p < 0.05). In contrast, tear meniscus volume in lens wearers increased on eye opening and recovered by 10 min later (p < 0.05). Tear meniscus volumes in CL wearers were less than those in controls at eye opening. NITBUT and comfort scores decreased immediately after CL insertion (p = 0.00). After eye opening, NITBUT was larger than the presleep levels until 10 min later. The comfort score was lower than the presleep values until 5 min later. CONCLUSIONS Dynamic changes in the tear menisci, NITBUT, and comfort level were evident in both inexperienced and experienced CL wearers during and after overnight wear. Decreased tear meniscus volumes and tear film stability may contribute to lower eye comfort for extended CL wearers. The tearing on eye opening may facilitate improvement of ocular comfort and tear refreshment for a reconditioning of the tear system and the CL.