Background: The aim of this study was to test the effects of cycling shoe outsole stiffness on both performance and comfort parameters during sub- and supra-maximal cycling tests. Methods: Two groups of recreational women tested three cycling shoe conditions with differing outsole stiffness. One group of 8 women performed four cycling tests of 3 min composed of two intensities (100 and 140 W) and two pedaling rates (70 and 100 rpm) for each pair of shoes. Metabolic and subjective perception of comfort measurement was evaluated with each shoe. Another group of 12 women performed 6-s all-out sprints against two external resistances (0.4 and 0.7 N/kg) to determine force-velocity relationships with the three cycling shoe conditions. Results: The main findings are that the stiffness of the investigated outsole cycling shoes (i) does not influence cycling performance whatever the test (ii) while the perception of comfort is largely degraded compared to the most flexible shoe. Conclusion: Maximizing stiffness should no longer be of the highest design principal for beginners or recreational women cyclists.