Purpose Digital or computerised eye charts are becoming standard in the examination of visual acuity. Each instrument allows the selection of different optotypes, presentation modalities, and crowding. The aim of this study was to examine the differences in visual acuity (VA) measurement using a digital eye chart, comparing different optotypes and procedures, together with an evaluation of the repeatability of the measurement. Methods Two groups of 52 participants aged between 18 and 31 years participated in the study. In the first experiment, VA thresholds were measured using LEA Symbols, Tumbling E, and Landolt Rings in monocular and binocular conditions using single line presentation and QUEST presentation. In the second experiment, we have compared all modalities of presentation together with a paper eye-chart and test the repeatability. Results The results showed that thresholds for LEA Symbols are low. The modality of presentation affects these thresholds. For Landolt Rings and Tumbling E, the QUEST procedure gave significantly better thresholds than line presentation, while this difference was absent for LEA Symbols. In comparing all modalities of presentation, single letter and line presentation showed similar values, slightly better than block presentation. Paper eye-charts showed better values of VA. Repeatability and agreement were good for all presentations, but best for QUEST. Conclusions The QUEST modality of presentation provides a better threshold than line presentation except for LEA Symbols. Examiners using digital eye charts must take into account that not all modalities of presentation and optotypes are equivalent and give different VA thresholds. Specific thresholds need to be used for each optotype and presentation modality.