PROJECT SUMMARY/ABSTRACT Over 50% of women in the U.S. are overweight or obese when they enter pregnancy. Women with obesity are 2.6 times more likely to experience lactation failure, independent of intention to breastfeed, contributing to adverse health outcomes for two generations. Inflammation, a metabolic hallmark of obesity, has been associated with lactation failure, but mechanistic studies evaluating the mechanisms underlying lactation failure in women are sparse. We now have a unique opportunity to address this knowledge gap through The Lifestyle Intervention in Preparation for Pregnancy (LIPP) study, a randomized controlled trial of a lifestyle (nutritional and exercise) intervention in women with overweight and class I obesity who are planning a subsequent pregnancy in the next two years. LIPP participants are recruited three months post-partum and after baseline metabolic measurements, are randomized to a closely supervised lifestyle intervention, consisting of caloric restriction with a shift to a Mediterranean diet pattern and exercise for 9-21 months, or standard care. The LIPP Lactation study seeks to understand the impact of this intervention on lactation outcomes and breastmilk composition. Our overall hypothesis is that a favorable post-partum metabolic environment has a durable impact on the metabolic health of two generations through longer lactation duration and altered breastmilk fatty acid composition. We will evaluate this hypothesis through the following specific aims: Aim 1. To quantify the effect of a post-partum lifestyle intervention on lactation duration and identify underlying mechanisms; Aim 2. To determine the impact of a lifestyle intervention and subsequent dietary and energy expenditure patterns on breastmilk fatty acid composition and infant growth; Exploratory Aim 3. To identify the impact of maternal lifestyle intervention on breastmilk metabolomic patterns. The post-partum period has been under-leveraged as a critical period to improve lifelong maternal and infant health. Scientifically, the LIPP Lactation study will fundamentally and rigorously inform our understanding of the mechanisms underlying lactation outcomes and the maternal predictors of breastmilk composition by leveraging the RCT design of the parent LIPP study. From a public health standpoint, LIPP Lactation provides a unique opportunity to identify discrete metabolic targets for intervention in women with obesity, to improve the lifelong health of two generations.