Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. An intensive use of the bicycle may increase the risk of erectile dysfunction and the compression of the perineal area has been showed to be a major mechanism leading to sexual alterations compromising the quality of life. Manufacturers claim that pads contribute to increase cyclists perineal protection ensuring a high level of comfort. To investigate the influence of various cycling pads with regard to perineal protection and level of comfort. Nine club road cyclists rode 20 min on a drum simulator, located at the Nutrition and Exercise Physiology Laboratory, at a constant speed and gear ratio wearing the shorts with 3 cycling pads of different design and thickness: basic (BAS), intermediate (INT), and endurance (END). Kinematics and pressure data were recorded at min 5, 15, and 20 of the test using a motion capture system and a pressure sensor mat. The variables of interest were: 3-dimensional pelvis excursions, peak pressure, mean pressure, and vertical force. The comfort level was assessed with a ranking order based on the subjects' perception after the 20-min trials and measuring the vertical ground reaction force under the anterior wheel as well as the length of the center of pressure (COP) trajectory on the saddle. Results showed that the vertical force and the average value of mean pressure on the saddle significantly decreased during the 20-min period of testing for BAS and END. Mean peak pressure on the corresponding perineal cyclist area significantly increased only for BAS during the 20-min period. Interestingly objective comfort indexes measured did not match cyclists subjective comfort evaluation. The lower capacity of BAS to reduce the peak pressure on the corresponding perineal area after 20 min of testing, together with its positive comfort evaluation, suggest that a balance between protection and perceived comfort should be taken into account in the choice of the pad. Hence, the quantitative approach of objective comfort indexes introduced in this study could be helpful for manufacturers in the development of their protective pads.