Human skeletal hemodynamics remain understudied. Neither assessments in weight-bearing bones during walking nor following periods of immobility exist, despite knowledge of altered nutrient-artery characteristics after short-duration unloading in rodents. We studied 12 older adults (8 females, aged 59 ± 3 years) who participated in ambulatory near-infrared spectroscopy (NIRS) assessments of tibial hemodynamics before (PRE) and after (POST) 14 days of head-down bed rest (HDBR), with most performing daily resistance and aerobic exercise countermeasures during HDBR. Continual simultaneous NIRS recordings were acquired over the proximal anteromedial tibial prominence of the right lower leg and ipsilateral lateral head of the gastrocnemius muscle during supine rest, walking, and standing. During 10 minutes of walking, desaturation kinetics in the tibia were slower (time to 95% nadir values 125.4 ± 56.8 s versus 55.0 ± 30.1 s, p = 0.0014). Tibial tissue saturation index (TSI) immediately fell (−9.9 ± 4.55) and did not completely recover by the end of 10 minutes of walking (−7.4 ± 6.7%, p = 0.027). Upon standing, total hemoglobin (tHb) kinetics were faster in the tibia (p textless 0.0001), whereas HDBR resulted in faster oxygenated hemoglogin (O2Hb) kinetics in both tissues (p = 0.039). After the walk-to-stand transition, changes in O2Hb (p = 0.0022) and tHb (p = 0.0047) were attenuated in the tibia alone after bed rest. Comparisons of NIRS-derived variables during ambulation and changes in posture revealed potentially deleterious adaptations of feed vessels after HDBR. We identify important and novel tibial hemodynamics in humans during ambulation before and after bed rest, necessitating further investigation.