The purposes of this pilot study were to explore the temperature changes of the paw pads in healthy cats before and after a 6-min walking test (6MWT), and to further compare the accuracy of the point and line region of interest (ROI) selection methods of the thermal imaging. Five healthy intact cats were recruited and trained to run on the treadmill, and to keep still during the standing phase on a glass panel when capturing thermographic imaging data. The paw pad temperature was recorded using a thermal camera and then analysed using both the point and line ROI before and after the 6MWT. The symmetry index (SI) between the limbs (left and right) was further calculated. Compared to the baseline, there were significant increases in the paw pad temperature of the forelimbs (point of left P < 0.001, point of right P = 0.048, point of average P = 0.002, line of left P < 0.001, line of right P = 0.007, line of average P < 0.001), hindlimbs (point of left P < 0.001, point of right P < 0.001, point of average P < 0.001, line of left P < 0.001, line of right P < 0.001, line of average P < 0.001), and average value after the 6MWT both using the point and line ROI selection methods. However, there is no temperature difference between the point and line ROI either before or after the 6MWT. In addition, no difference in the SI was found between the baseline and post-test or between the point and line ROI. These results preliminarily indicated that a 6WMT and thermal imaging could be a good combination for further clinical practices to recognise lameness or any other gait disabilities in cats, and both the point and line ROI selection methods can be considered when analysing thermographic data.