During acute altitude exposure tachycardia increases cardiac output (Q) thus preserving systemic O2 delivery. Within days of acclimatization, however, Q normalizes following an unexplained reduction in stroke volume (SV). To investigate whether the altitude-mediated reduction in plasma volume (PV) and hence central blood volume (CBV) is the underlying mechanism we increased/decreased CBV by means of passive whole body head-down (HDT) and head-up (HUT) tilting in seven lowlanders at sea level (SL) and after 25/26 days of residence at 3454 m. Prior to the experiment on day 26, PV was normalized by infusions of a PV expander. Cardiovascular responses to whole body tilting were monitored by pulse contour analysis. After 25/26 days at 3454 m PV and blood volume decreased by 9 ± 4% and 6 ± 2%, respectively (P < 0.001 for both). SV was reduced compared to SL for each HUT angle (P < 0.0005). However, the expected increase in SV from HUT to HDT persisted and ended in the same plateau as at SL, albeit this was shifted 18 ± 20° toward HDT (P = 0.019). PV expansion restored SV to SL during HUT and to an ∼8% higher level during HDT (P = 0.003). The parallel increase in SV from HUT to HDT at altitude and SL to a similar plateau demonstrates an unchanged dependence of SV on CBV, indicating that the reduced SV during HUT was related to an attenuated CBV for a given tilt angle. Restoration of SV by PV expansion rules out a significant contribution of other mechanisms, supporting that resting SV at altitude becomes reduced due to a hypovolemia.