Dehydration reduces flexibility and increases stiffness in male collegiate age runners. Dehydration has been shown to negatively affect collagen in vitro; however the literature lacks works exploring the in vivo effects of dehydration on collagenous tissue. This study addresses this gap in the literature, by exploring the effects of dehydration on the muscles and connective tissues of the posterior leg. It was hypothesized that when dehydrated, the collagen within these tissues would become stiffer, decreasing flexibility and increasing stiffness. A cross-over cohort design was conducted to evaluate nineteen male collegiate runners. Each subject attended three sessions: baseline, dehydration and euhydration. The order of testing was randomly assigned and the PI was blinded throughout. Mean sit and reach (MSnR), mean terminal straight leg raise (MTSLR) and mean posterior leg stiffness (MPLS) scores for each testing condition were analyzed using a repeated measures ANOVA. Dehydrated, subjects demonstrated statistically significant decreases in MSnR scores, p<0.001, d=0.469 (MSnR dehydrated 26.83 ± 7.53 cm and MSnR euhydrated 30.36 ± 7.53 cm) and MTSLR, p<0.001, d=1.068 (MTSLR dehydrated 51.38 ± 9.39 and MTSLR euhydrated 60.58 ± 7.74), with a concurrent increase in MPLS, p=0.005, d=1.023 (MPLS dehydrated 0.899 ± 0.357 and MPLS euhydrated 0.508 ± 0.409), as compared to when they were euhydrated. The large effect size for MPLS and MTSLR and moderate for MSnR indicates that when dehydrated subjects became stiffer and has less flexibility as compared to when they are euhydrated. These changes may impede performance and increase the risk of injury in dehydrated individuals.