We evaluated the effects of fish oil and/or probiotic supplementation in a randomized placebo-controlled intervention pilot trial on gestational weight gain (GWG) and body composition. Additionally, the influence of gestational diabetes (GDM) on GWG and body composition was assessed. We randomized 439 overweight or obese women (mean 13.9±2.1 gestational weeks) into four intervention groups: fish oil+placebo, probiotics+placebo, fish oil+probiotics and placebo+placebo. Fish oil (1.9g docosahexaenoic acid and 0.22g eicosapentaenoic acid) and probiotic supplements (Lactobacillus rhamnosus HN001 and Bifidobacterium animalis ssp. lactis 420, 1010 CFU each) were consumed daily from randomization until delivery. GDM was diagnosed with 2-hour 75g oral glucose tolerance test. Body composition was measured with air displacement plethysmography at randomization and in late pregnancy (mean 35.2±0.9 gestational weeks). Fish oil and/or probiotic intervention did not influence mean GWG or change in body fat mass or body fat percentage of the women (p>0.17 for all comparisons). Body composition in early pregnancy did not differ between the women who did or did not develop GDM (adjusted p>0.23). Compared to the normoglycemic women (n=278), women diagnosed with GDM (n=119) gained less weight (7.7±0.4kg vs. 9.3±0.4kg, adjusted mean difference -1.66 [-2.52, -0.80],p<0.001) and fat mass (0.4±0.4kg vs. 1.8±0.3kg, adjusted mean difference -1.43 [-2.19, -0.67],p<0.001) during the follow-up. In conclusion, adiposity of pregnant overweight and obese women was not affected by supplementation with fish oil and/or probiotics, nor did it predict the development of GDM. However, adiposity was reduced in women with GDM compared to normoglycemic women irrespective of the nutritional intervention.