Numerous studies that have investigated distal femoral cartilage thickness before and after a prescribed loading activity have included a rest period prior to the loading activity to remove the effects of previous activities that may have compressed the cartilage. The purpose of this study was to systematically determine how unloading time affects cartilage thickness, as well as how sex and leg preference influence the thickness in young, active adults with no knee injuries. Thirty-two healthy college students (16 female, 16 male) who regularly participated in moderate or vigorous physical activity were recruited and instructed to refrain from any physical activity at least 12 h prior to data collection. Ultrasound images were initially obtained for each leg and then every 15 min for an hour (T0, T15, T30, T45, T60) using a Mindray 7 machine. Between collections, they had their legs extended in front of them. Their leg preference was determined using the Waterloo Footedness Questionnaire. Cartilage thickness was measured at the intercondylar notch (IC), medial condyle (MC), and lateral condyle (LC). The results suggest that the cartilage at the LC experiences less compression than the other regions. Among individuals who have reduced their physical activity for at least 12 h, resting with legs extended for 30 min appears to allow the IC and MC cartilage to recover and create equivalent conditions for all participants. While female values were lower than those of males across all regions, there was no difference in their response to unloading.