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OBJECTIVE To investigate the effects of a small sensor attached to the pastern region of a forelimb on lameness detection and quantification with a portable inertial sensor–based system (PISBS) for lameness detection and quantification in horses. ANIMALS 20 adult horses (body weight, 410 to 650 kg) with no visible lameness at the walk. PROCEDURES In a crossover study design, horses were evaluated at the trot twice using the PISBS with the gyroscope alternately attached to the right forelimb pastern region (as recommended by the manufacturer) or to the left forelimb pastern region (with the sensor flipped 180° on the frontal plane relative to the standard position). Agreement between the 2 instrumentation approaches was investigated graphically and by repeated-measures ANOVA, Pearson correlation analysis, and Bland-Altman analysis. RESULTS No effects of instrumentation of a forelimb with the gyroscope were detected on the lame limb or limbs or on lameness severity. Attachment of the gyroscope to a forelimb had no effect on forelimb or hind limb lameness (ie, did not cause or mask lameness) as measured with the PISBS. CONCLUSIONS AND CLINICAL RELEVANCE Instrumentation of a forelimb with a lightweight gyroscope for lameness evaluations with a PISBS had no effects on lameness measurements in horses. Results suggested that, when indicated, the gyroscope can be attached (while flipped 180° on the frontal plane relative to the standard position) to the left forelimb (rather than the right forelimb).