This study tested the hypothesis that prolonged physical deconditioning affects the coupling of left ventricular depolarization to its ejection (the pre-ejection period, PEPi) and that this effect is minimized by exercise countermeasures. Following assignment to non-exercise (Control) and exercise groups (Exercise), 14 females performed 56 days of continuous head-down tilt bed rest. Measurements of the electrocardiogram (ECG) and stroke volume (Doppler ultrasound) during supine rest were obtained at baseline prior to (Pre) and after (Post) the head-down tilt bed rest (HDBR) period. Compared with Pre, the PEPi was increased following head-down tilt bed rest (main effect, P < 0.005). This effect was most dominant in the Control group [Pre = 0.038 ± 0.06 s (s.d.) versus Post = 0.054 ± 0.011 s; P < 0.001]. In the Exercise group, PEPi was 0.032 ± 0.005 s Pre and 0.038 ± 0.018 s Post; P= 0.08. Neither the QRS interval nor cardiac afterload was modified by head-down tilt bed rest in Control or Exercise groups. Low-dose isoprenaline infusion reversed the head-down tilt bed rest-induced delay in the PEPi. These results suggest that head-down tilt bed rest leads to a delayed onset of systolic ejection following left ventricular depolarization in a manner that is affected little by the exercise countermeasure but is related to β-adrenergic pathways. The delayed onset of systole following head-down tilt bed rest appears to be related to mechanism(s) affecting contraction of the left ventricle rather than its depolarization.