To prospectively compare laser in situ keratomileusis (LASIK) flaps created with a femtosecond laser or a mechanical microkeratome and analyze the effect of the postoperative measurement interval on estimated femtosecond flap thickness using ultrasound (US) pachymetry. Department of Ophthalmology, University Hospital of Coimbra, and Coimbra Surgical Centre, Coimbra, Portugal. Flaps were created with a Hansatome Zero Compression microkeratome (Group 1), Zyoptix XP keratome (Group 2), or IntraLase 60 kHz femtosecond laser (Groups 3 and 4). Flap thickness was determined by intraoperative US pachymetry immediately after flap creation in Groups 1, 2, and 3. In Group 4, pachymetry was performed 20 minutes after the laser treatment. The main outcome measures were flap thickness and deviation from the target value. Eighty patients had LASIK for myopia or myopic astigmatism. The mean flap thickness was 149.1 microm +/- 24.9 (SD) in Group 1, 124.7 +/- 23.8 microm in Group 2, 143.1 +/- 18.4 microm in Group 3, and 115.5 +/- 12.5 microm in Group 4. The difference in flap thickness between Group 3 and Group 4 was statistically significant (P<.01). The flap thickness deviation from the target value was 22.8 microm in Group 1, 19.0 microm in Group 2, 26.1 microm in Group 3, and 10.4 microm in Group 4. Results indicate that the time of measurement after femtosecond affects the estimated flap thickness. Waiting 20 minutes after laser treatment permitted easier separation and may eliminate the effect of variable corneal dehydration on flap measurement by subtraction pachymetry.