Background Obesity is an increasing problem worldwide and is associated with serious health risks. Obesity not only reduces physical health, but can also negatively affect levels of perceived stress, mood symptoms, sleep quality and quality of life (QoL), which may lead to further weight gain. We have previously shown that a pre-conception lifestyle intervention reduced weight and improved physical QoL in the short term. In the current study, we assessed the effects of this intervention in women with obesity and infertility on perceived stress, mood symptoms, sleep quality and QoL five years after randomization. Methods and findings We followed women who participated in the LIFEstyle study. This is a multi-center randomized controlled trial comparing a six-month lifestyle intervention to improve diet and increase physical activity followed by infertility treatment, versus prompt infertility treatment. Participants were 577 women with infertility between 18 and 39 years of age with a body mass index (BMI) 29 kg/m 2 . For the current study we measured perceived stress, mood symptoms, sleep quality and QoL in 178 women five years after randomization. T-tests and linear regression models were used to assess differences between the intervention and control groups. Five years after randomization, no differences were observed for perceived stress, mood symptoms, sleep quality and QoL between the intervention (n = 84) and control groups (n = 94). There was selective participation: women who did not participate in the follow-up had lower baseline mental QoL, and benefitted more from the intervention in terms of improved physical QoL during the original LIFEstyle intervention. Conclusions We found no evidence that a pre-conception lifestyle intervention improved female well-being five years after randomization.