The objective of the in vitro experiments described in this paper was to quantify the effects of some instrumental variables on the quantitative evaluation, by means of first-order gray-level statistics, of ultrasonographic images of equine tendons. The experiments were done on three isolated equine superficial digital flexor tendons that were mounted in a frame and submerged in a waterbath. Sections with either normal tendon tissue, an acute lesion, or a chronic scar, were selected. In these sections, the following experiments were done: 1) a gradual increase of total amplifier gain output subdivided in 12 equal steps; 2) a transducer tilt plus or minus 3 degrees from perpendicular, with steps of 1 degree; and 3) a transducer displacement along, and perpendicular to, the tendon long axis, with 16 steps of 0.25 mm each. Transverse ultrasonographic images were collected, and in the regions of interest (ROI) first-order gray-level statistics were calculated to quantify the effects of each experiment. Some important observations were: 1) the total amplifier gain output has a substantial influence on the ultrasonographic image; for example, in the case of an acute lesion, a low gain setting results in an almost completely black image; whereas, with higher gain settings, a marked "filling in" effect on the lesion can be observed; 2) the relative effects of the tilting of the transducer are substantial in normal tendon tissue (18%) and chronic scar (12%); whereas, in the event of an acute lesion, the effects on the mean gray level are dramatic (40%); and 3) the relative effects of displacement of the transducer are small in normal tendon tissue, but on the other hand, the mean gray-level changes 7% in chronic scar, and even 20% in an acute lesion. In general, slight variations in scanner settings and transducer handling can have considerable effects on the gray levels of the ultrasonographic image. Furthermore, there is a strong indication that this quantitative method, as far as based exclusively on the first-order gray-level statistics, may be not discriminative enough to accurately assess the integrity of the tendon. Therefore, the value of a quantitative evaluation of the first-order gray-level statistics for the assessment of the integrity of the equine tendon is questionable.