There remains uncertainty regarding the magnitude and patterns of variability in some aspects of physiology in normal pregnancy. Previous work has indicated an influence of gestation on the autonomic nervous system (ANS) control of heart rate but a definitive study of this is beneficial (Moertl et al., 2009). Characterising and understanding such changes during normal pregnancy could benefit in early identification of abnormal physiological function and identify patients at risk for adverse responses to interventions such as vasodilator therapy (Walther et al., 2005). Longitudinal study designs involving cardiovascular reflex tests during antenatal and post-partum periods are likely to yield information on the dynamic adaptations to pregnancy. We sought to obtain a longitudinal characterisation of the influence of normal pregnancy on ANS function using heart rate variability (HRV) analysis. Continuous ECG monitoring (Reynolds Holter; Spacelabs Medical UK) assessed autonomic function in a group of 51 healthy non-pregnant (NP) females (20-40 yrs). Longitudinal assessment was then performed on healthy pregnant women (20-40 yrs) at 14.8±0.4 weeks (T1, n=32), 25.9±0.2 weeks (T2, n=20), 35.9±0.7 weeks (T3, n=14) and at 12.3±0.7 weeks post partum (PP, n=7). Participants performed a protocol that consisted of posture (supine 6 min, standing 6 min), step exercise (6 min), seated recovery (6 min), mental arithmetic (3 min), metronomic breathing (3 min) and spontaneous breathing (3 min). R-R interval (RRI), RMSSDRR, SDRR, LF, HF and Total Power were investigated via repeated measures ANOVA which assessed the influence of trimester, protocol stage and their interaction (pregnancy status x stage). One-way ANOVA additionally compared NP and trimester at each specific stage of the protocol. Significant difference in trimester and stage interaction were found in LFn RR (P= 0.25) and RRI (P=0.03). Comparing responses over the whole protocol, there was a significant influence of pregnancy-stage on HFn (P=0.03), HR (P=0.04) and RRI (P=0.01), attributable to T1 vs T3 differences. Significant changes were also found in RMSSDRR (P=0.03) and RRI (P=0.03), between T3 vs PP. A significant influence of trimester (P <0.05) was evident in the responses to ‘postural change’ (supine-to-standing) and respiratory frequency change (spontaneous-to-metronomic breathing) between the paired groups NP vs T3, T1 vs T3 and T3 vs PP. Our protocol was designed to provoke autonomic nervous system responses and was assessed via changes in HRV. Non-pregnant responses were similar to those during T1 and response trends were independent of pregnancy status. However, we observed a significant influence of advanced gestation on the postural and respiratory-induced responses compared with NP and T1 pregnancy groups. We will extend this pilot work to use this protocol to developing a screening tool for antenatal disorders.