BackgroundCardiovascular disease, including hypertension, in pregnant women is a leading cause of morbidity and mortality globally. The development of reference intervals for cardiovascular responses using exercising testing to measure oxygen utilisation (V̇O2) with cardiopulmonary exercise testing (CPET), and distances walked using the incremental shuttle walk test (ISWT), may be promising methods to assess and stratify pregnant women regarding their risk of adverse pregnancy outcomes, to encourage exercise during pregnancy, and to improve exercise prescriptions during pregnancy. We aimed to determine the reference intervals for V̇O2 at rest, anaerobic threshold (AT) and submaximal exercise using CPET, and the reference interval for the ISWT, to develop a correlation equitation that predicts submaximal V̇O2 from the distance walked in the ISWT, and to explore the relationship between hemoglobin (Hb) and ferritin concentration and V̇O2 at AT in women in second trimester. MethodsAfter prospective IRB approval (HREC 15/23) and clinical trials registration (ANZCTR ACTRN12615000964516), and informed written consent, we conducted CPET and the ISWT according to international guidelines in a university associated tertiary referral obstetric and adult medicine hospital, in healthy pregnant women in second trimester (14 to 27 gestational weeks). Hemoglobin and ferritin concentrations were recorded from pathology results in the participants’ medical records at the time of exercise testing. Adverse events were recorded. Results90 participants undertook CPET, 28 of which also completed the ISWT. The mean ± SD age and body mass index (BMI) were 32 ± 3.2 years, and 25 ± 2.7 kg/m2. Median (IQR) gestation was 23 (22,24) weeks. One in four women were 24 weeks or greater gestation. The reference intervals for V̇O2 at rest, AT and submaximal exercise were 2.9 to 5.3, 8.1 to 20.7 and 14.1 to 30.5 mL/kg/min respectively. The reference interval for the ISWT was 218 to 1058 meters.metres. The correlation equation to predict submaximal V̇O2 from the distance walked in the ISWT was submaximal V̇O2 (mL/kg/min) = 0.012*distance walked in ISWT (m) + 14.7 (95% CI slope 0.005 to 0.070, Pearson r = 0.5426 95% CI 0.2126 to 0.7615, P=0.0029). Hemoglobin concentration was positively correlated with V̇O2 at AT (AT V̇O2 (mL/kg/min) = 0.08*Hb (g/L) + 4.9 (95% CI slope 0.0791 to 0.143, Pearson r = 0.2538 95% CI 0.049 to 0.438, P=0.016). There was no linear association between ferritin and submaximal V̇O2 (Pearson r = 0.431 P=0.697). There were no maternal or fetal complications. ConclusionsCPET and ISWT are safe and feasible in women in second trimester including those at or beyond 24 weeks gestation. We have established the reference interval for V̇O2 at rest, AT and submaximal exercise by CPET, the reference interval for the distance walked for the ISWT, and a correlation equation to predict submaximal V̇O2 for use in clinical practice and research. Hemoglobin rather than ferritin is likely correlated with exercise capacity in pregnancy suggesting vigilance to correct lower hemoglobin levels may positively impact maternal health. Clinical trials registryThe study was prospectively registered with the Australian and New Zealand Clinical Date of registration – 15/9/2015; Date of initial participant enrolment - 4/11/2015; Clinical trial identification number; ACTRN12615000964516; URL of the registration site - https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=369216 Prior presentation and published abstract“Oxygen utilization and anaerobic threshold measured using cardiopulmonary exercise testing in healthy pregnant women.” Obstetric Anaesthetists Association Meeting Edinburgh May 2023 Traer E, Ismail H, Leeton L, Bernhard R, Dennis AT. Oxygen utilisation and anaerobic threshold measured using cardiopulmonary exercise testing in healthy pregnant women. International Journal of Obstetric Anesthesia 2023;54: Supplement 1. Article 103659 O7 Traer E, Ismail H, Leeton L, Bernhard R, Dennis AT. “Oxygen utilization and anaerobic threshold measured using cardiopulmonary exercise testing in healthy pregnant women.” Poster presentation, iPOETTS abstract competition. Evidence Based Perioperative Medicine EPBOM World Congress of Perioperative Medicine, July 2023, London, U.K.