Objective To assess the association between marked variability in fetal heart rate (FHR) and neonatal acidosis. Design Bicentric prospective cohort study. Setting From January 2019 to December 2019, in two French tertiary care maternity units. Population Women in labour at ≥37 weeks of gestation, with continuous FHR monitoring until delivery and with the availability of umbilical arterial pH. Women with intrauterine fetal death or medical termination, multiple pregnancies, non-cephalic presentation or planned caesarean delivery were excluded. Methods The exposure was marked variability in FHR in the 60 minutes before delivery, defined as a variability greater than 25 beats per minute, with a minimum duration of 1 minute. To assess the association between marked variability and neonatal acidosis, we used multivariable modified Poisson regression modelling. We then conducted subgroup analyses according to the US National Institute of Child Health and Human Development (NICHD) category of the associated fetal heart rate. Main outcome measures Neonatal acidosis, defined as an umbilical artery pH of ≤7.10. Results Among the 4394 women included, 177 (4%) had marked variability in fetal heart rate in the 60 minutes before delivery. Acidosis occurred in 6.0% (265/4394) of the neonates. In the multivariable analysis, marked variability was significantly associated with neonatal acidosis (aRR 2.30, 95% CI 1.53–3.44). In subgroup analyses, the association between marked variability and neonatal acidosis remained significant in NICHD category-I and category-II groups. Conclusions Marked variability was associated with a twofold increased risk of neonatal acidosis.