Purpose: Orthostasis at sea level decreases brain tissue oxygenation and increases risk of syncope. High altitude reduces brain and peripheral muscle tissue oxygenation. This study determined the effect of short-term altitude acclimatization on cerebral and peripheral leg tissue oxygenation index (TOI) post-orthostasis. Method: Seven lowlanders completed a supine-to-stand maneuver at sea level (450 m) and for 3 consecutive days at high altitude (3,776 m). Cardiorespiratory measurements and near-infrared spectroscopy-derived oxygenation of the frontal lobe (cerebral TOI) and vastus lateralis (leg TOI) were measured at supine and 5 min post-orthostasis. Results: After orthostasis at sea level, cerebral TOI decreased (mean Δ% [95% CI]: -4.5%, [-7.5, -1.5], P < 0.001) whilst leg TOI was unchanged (-4.6%, [-10.9, 1.7], P = 0.42). High altitude had no effect on cerebral TOI following orthostasis (day 1 to 3: -2.3%, [-5.3, 0.7]; -2.4%, [-5.4, 0.6]; -2.1%, [-5.1, 0.9], respectively, all P > 0.05) whereas leg TOI decreased (day 1 to 3: -12.0%, [-18.3, -5.7]; -12.1%, [-18.4, -5.8]; -10.2%, [-16.5, -3.9], respectively, all P < 0.001). This response did not differ with days spent at high altitude, despite evidence of cardiorespiratory acclimatization (increased peripheral oxygen saturation [supine: P = 0.01; stand: P = 0.02] and decreased end-tidal carbon dioxide [supine: P = 0.003; stand: P = 0.01]). Conclusion: Cerebral oxygenation is preferentially maintained over leg oxygenation post-orthostasis at high altitude, suggesting different vascular regulation between cerebral and peripheral circulations. Short-term acclimatization to high altitude did not alter cerebral and leg oxygenation responses to orthostasis.