Consensus guidelines have attempted to standardize the measurement and interpretation of pulse wave velocity (PWV); however, guidelines have not addressed whether hydration status affects PWV. Moreover, multiple studies have utilized heat stress to reduce arterial stiffness which may lead to dehydration. This study utilized two experiments to investigate the effects of dehydration on PWV at rest and during passive heat stress. In experiment 1, subjects (n = 19) completed two trials, one in which they arrived euhydrated and one dehydrated (1·2[1·0]% body mass loss). In experiment 2, subjects (n = 11) began two trials euhydrated and in one trial did not receive water during heat stress, thus becoming dehydrated (1·6[0·6]% body mass loss); the other trial subjects remained euhydrated. Using Doppler ultrasound, carotid-to-femoral (central) and carotid-to-radial (peripheral) PWVs were measured. PWV was obtained at a normothermic baseline, and at a 0·5°C and 1°C elevation in rectal temperature (via passive heating). In experiment 1, baseline central PWV was significantly higher when euhydrated compared to dehydrated (628[95] versus 572[91] cm s , respectively; P<0·05), but peripheral PWV was unaffected (861[117] versus 825[149] cm s ; P>0·05). However, starting euhydrated and becoming dehydrated during heating in experiment 2 did not affect PWV measures (P>0·05), and independent of hydration status peripheral PWV was reduced when rectal temperature was elevated 0·5°C (-74[45] cm s ; P<0·05) and 1·0°C (-70[48] cm s ; P<0·05). Overall, these data suggest that hydration status affects measurements of central PWV in normothermic, resting conditions. Therefore, future guidelines should suggest that investigators ensure adequate hydration status prior to measures of PWV.