To evaluate the learning effect of Frequency Doubling Technology (FDT) perimetry using the Humphrey Matrix-FDT perimetry (Matrix) 24-2 full-threshold program on patients with ocular hypertension experienced with standard automated perimetry. Twenty-four patients with ocular hypertension underwent 5 full-threshold Matrix tests at intervals of 5+/-2 days. Learning effect was defined as an improvement at results for duration, perimetric indices, foveal sensitivity, Glaucoma Hemifield Test, and the number of points with a P<5% and <1% in the total and pattern deviation maps. Eccentricity, hemifield, and quadrant sensitivities were also addressed as sources of differences in learning effect. Test-retest variability was also calculated for each repetition as the mean of the point-to-point interindividual standard deviations. A learning effect was demonstrated for mean defect (P=0.031, analysis of variance) and foveal sensitivity (P=0.009) and it only affected the first test for both parameters. All the other parameters did not show any significant learning effect. The effect was independent from eccentricity and quadrant or hemifield sensitivities. The results of this study demonstrate that the learning effect for Matrix-FDT is mild and it may affect only the first test. Caution is needed in the analysis of the first Matrix-FDT examination and retest may be advisable in the presence of low mean defect.