AIM:To evaluate the effect of misalignment on the measurements of retinal nerve fiber layer (RNFL) by spectral-domain optical coherence tomography (OCT).METHODS:A total of 42 eyes from 21 healthy young subjects underwent RNFL measurements with RTVue spectral-domain OCT (Optovue Inc., Fremont, California, USA). Two baseline measurements with perfectly aligned central circle to the borders of the optic nerve and four misaligned measurements which were misaligned towards to four quadrants were taken. The differences in RNFL between the baseline and misaligned measurements were analyzed with a new algorithm called Helvacioglu reproducibility index (HRI) which is designed to measure the reproducibility of the scans by evaluating the RNFL changes in the four main quadrants.RESULTS:The average RNFL scores of the first two baseline measurements have good correlation (c=0.930) and good reproducibility scores (0.15±0.07). Superior misaligned measurements had significantly lower superior quadrant score and higher inferior quadrant score, similar nasal and little higher temporal scores (P1, P2<0.001, P3=0.553, P4=0.001). Inferior misaligned measurements had significantly higher superior quadrant score and lower inferior quadrant score with similar temporal and little lower nasal scores (P1, P2<0.001, P3=0.315, P4=0.016). Nasal misaligned measurements had significantly higher temporal quadrant score and lower nasal quadrant score with little lower superior and inferior scores (P1, P2, P4<0.001, P3=0.005). Temporal misaligned measurements had significantly higher nasal quadrant score and lower temporal quadrant score with similar superior and little higher inferior scores (P1, P2<0.001, P3=0.943, P4=0.001).CONCLUSION:Good alignment of the central circle to the borders of optic nerve is crucial to have correct and repeatable RNFL measurements. Misalignment to a quadrant resulted in falsely low readings at that quadrant and falsely high readings at the opposite quadrant.