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Introduction: Physical activity (PA) during pregnancy has numerous benefits, which may be mediated via effects on the immune system. However, supportive evidence is inconsistent and is mainly from studies in high-risk groups. We estimated the effect of PA during pregnancy on systemic inflammatory markers and cytokines in mothers recruited in the Barwon infant study., Material and Methods: The Barwon infant study is a prebirth cohort of 1064 mothers recruited in the Barwon Region of Victoria, Australia. Participants reported their previous week's PA at their 28-week antenatal appointment using the International PA Questionnaire. Women were grouped into low, moderate, and high PA categories based on daily duration and weekly frequency of walking, moderate- or vigorous-intensity PA. Women reporting moderate levels of PA, consistent with current recommendations, served as the comparison group. Markers of systemic inflammation, high-sensitivity C-reactive protein (hsCRP), glycoprotein acetyls (GlycA), and 17 cytokines were measured at 28 weeks gestation and log transformed as appropriate. Regression analyses adjusted for maternal smoking, gestational diabetes mellitus, prepregnancy BMI, and household size were performed., Results: Compared to women in the moderate group (n = 371, 42%), women reporting low PA (n = 436, 50%) had 10.1% higher hsCRP (95% CI (3.7% to 16.6%), p < 0.01) while women in high PA (n = 76, 9%) had a 14% higher hsCRP (95% CI (3.1% to 24.8%), p = 0.01). Women in the high PA category had higher interleukin (IL)-4 (q = 0.03) and IL-9 (q = 0.03) levels compared to those in moderate category. Each vigorous MET minute/week was associated with lower GlycA (β = -0.004, 95% CI (-0.044 to 0.035); p = 0.03)., Conclusions: Low and high PA are each associated with higher hsCRP than moderate PA, suggesting that undertaking the recommended moderate PA during pregnancy decreases systemic inflammation. High PA affects T cell-associated cytokines during pregnancy. Evidence from our study suggests that PA can modulate the immune responses during pregnancy. Studies are now required to assess whether PA during pregnancy impacts maternal and infant clinical outcomes by modifying inflammatory responses., (© 2024 The Author(s). Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)