The aim of the study was to define and characterize the optical behavior of the tear film during visual fixation in humans on en-face optical coherence tomography (OCT). We included 20 healthy participants, 60% female, aged from 25 to 42 years (33.05 ± 4.97 [mean ± SD]) and ten patients with severe dry eye, 50% female, aged from 26 to 42 years (33.7 ± 5.31). To perform high-resolution tear film imaging, participants were asked to gaze at the internal fixation point in the spectral-domain anterior segment OCT device, and meanwhile scanning session was executed at the following time-points after blinking: at the 2nd, 3rd, 4th, 5th, and 6th second. After one hour, OCT imaging was repeated (second session) by a different operator masked to the study to verify the reliability of results. During each measuring session, a pulse oximetry was used for continuously measuring the heart rate and oxygen saturation (SpO2%). A preliminary experiment was also performed to test the absence of geometric patterns from the anterior surface of a motionless artificial eye. OCT imaging showed a motionless, stable anterior surface of the artificial eye and in dry eye patients. Conversely, in the healthy participants of the study, a bull's eye pattern of the tear film was detected by OCT at the 2nd, 3rd, 4th, 5th, and 6th second after blinking, respectively, in 45%, 60%, 45%, 60%, and 40% of OCT scans during the first session, and in 35%, 65%, 65%, 60%, and 35% of cases in the second session. Overall, a total of 200 OCT scans were performed in normal human population. A significant correlation was found between the novel tear film pattern and heart rate during the first and the second session (p < 0.01) in healthy eyes. Conversely, no correlation was revealed with SpO2%. Intraclass correlation (ICC) analysis for OCT imaging of the tear film revealed a statistically significant reproducibility of the results (ICC = 0.838; p < 0.01), indicating the high level of reliability of the method, independently of heart rate and SpO2% variables. There exists a novel, geometric pattern of the tear film during visual fixation detectable by en-face OCT, which is mainly evident as heart rate increases. Its discovery implies in turn the presence of a specific vibration (or imperceptible motion) of the tear film that, at present, is not recognized and corrected by the OCT software (in image postprocessing) unlike other eyeball movements.