Human spaceflight removes the daily gravitational stress placed on the cardiovascular system and greatly reduces the normal levels of physical activity. Both of these factors could lead to cardiovascular deconditioning and increased risk of orthostatic hypotension on return to Earth. The spaceflight experiment “BP Reg” was designed to investigate the primary hypothesis that an inflight challenge to BP regulation by rapid deflation of thigh cuffs could be used as a test of inflight arterial baroreflex response to a sudden reduction in arterial blood pressure. Eight male astronauts provided written informed consent on a project approved by five independent Ethics Review Boards (including NASA MPA 7116301606HR). They then completed three repetitions in the pre-flight (supine position) and in the inflight condition of rapid deflation of two cuffs (Leg-Arm Cuff System) placed on the upper thighs for 3-min to pressures above systolic BP. Finger arterial pressure was recorded throughout the leg cuff tests (inflight by the Continuous Blood Pressure Device) and Modelflow cardiac output (QMF) was calculated. In addition, a rebreathing measurement of cardiac output was conducted to correct the QMF estimates. The change in QMF underestimated the change in cardiac output by rebreathing: no change was seen for QMF while rebreathing increased 47%. With release of the leg cuffs, there were no significant differences in the drops in systolic BP (16.4 ± 3.7 and 16.4 ± 5.6 mmHg, pre-flight to inflight) and in diastolic BP (15.7 ± 3.4 and 14.4 ± 3.2 mmHg), the increases in heart rate (18.6 ± 7.2 and 16.4 ± 3.3 bpm) and QMFcorrected (2.4 ± 1.0 and 2.5 ± 0.9 L/min). However, there was a greater drop in calculated total peripheral resistance pre-flight (8.9 ± 2.3 vs. 5.7 ± 0.9 units). The results showed that the leg cuffs were effective in spaceflight in causing a reduction in arterial blood pressure that activated the arterial baroreflex response, and that the heart rate component of the arterial baroreflex was unchanged from pre-flight. However, the responses were biased by the large increase in cardiac output with spaceflight. It is currently unknown where the 47% increase in cardiac output is directed during spaceflight but an hypothesis is that it goes to the splanchnic circulation and acts as a buffer for blood pressure regulation during a challenge such as the rapid change in total peripheral resistance with leg cuff release. The results suggest that the arterial baroreflex is not changed during spaceflight. The blood pressure response to other challenges during spaceflight such as lower body negative pressure should be assessed.