Background: It was the aim of this study to compare the repeatability of Spectralis optical coherence tomography (OCT) retinal thickness measurements in healthy subjects and diabetic patients with clinically significant macular edema (CSME) with or without the use of the follow-up system. Methods: Thirty-eight eyes of 38 healthy subjects (control group) and 68 eyes of 68 diabetic patients with CSME were included in the study. The coefficient of repeatability (CR) and intrasession coefficients of variation were tested with 20 × 15 degree raster scans consisting of 19 high-resolution line scans (15 frames per scan) that were repeated 3 times by 1 experienced examiner. The first scan was set as the reference scan, whereas the second and third scans were the follow-up scans and were performed with and without the use of the follow-up mode, respectively. Results: The means and standard deviations for the central foveal subfield (CSF) in healthy subjects and diabetic patients were 289 ± 21 and 402 ± 105 μm, respectively. Particularly in diabetic patients, examining the CSF, CR was 2.67% (10.73 µm) and 6.73% (27.01 µm) with and without using the follow-up mode, respectively, and the difference was statistically significant (p < 0.05). Conclusion: These results support the hypothesis that the follow-up system improves the repeatability either in healthy subjects or in diabetic patients with poor fixation. The wider improvement in repeatability in diabetic patients in the follow-up system group compared to the no follow-up system group are probably related to poor patient fixation or eye movement in patients with CSME.