Abstract Background Physical inactivity and overweight are two known risk factors for postmenopausal breast cancer. Former exercise intervention studies showed that physical activity influences sex hormone levels, known to be related to postmenopausal breast cancer, mainly when concordant loss of body weight was achieved. The question remains whether there is an additional beneficial effect of physical activity when weight loss is reached. The aim of this study is to investigate the effect attributable to exercise on postmenopausal breast cancer risk biomarkers, when equivalent weight loss is achieved compared with diet-induced weight loss. Design The SHAPE-2 study is a three-armed, multicentre trial. 243 sedentary, postmenopausal women who are overweight or obese (BMI 25–35 kg/m 2 ) are enrolled. After a 4-6 week run-in period, wherein a baseline diet is prescribed, women are randomly allocated to (1) a diet group, (2) an exercise group or (3) a control group. The aim of both intervention groups is to lose an amount of 5–6 kg body weight in 10–14 weeks. The diet group follows an energy restricted diet and maintains the habitual physical activity level. The exercise group participates in a 16-week endurance and strength training programme of 4 hours per week. Furthermore, they are prescribed a moderate caloric restriction. The control group is asked to maintain body weight and continue the run-in baseline diet. Measurements include blood sampling, questionnaires, anthropometrics (weight, height, waist and hip circumference), maximal cycle exercise test (VO 2peak ), DEXA-scan (body composition) and abdominal MRI (subcutaneous and visceral fat). Primary outcomes are serum levels of oestradiol, oestrone, testosterone and sex hormone binding globulin (SHBG). Discussion This study will give insight in the potential attributable effect of physical activity on breast cancer risk biomarkers and whether this effect is mediated by changes in body composition, in postmenopausal women. Eventually this may lead to the design of specific lifestyle guidelines for prevention of breast cancer. Trial registration The SHAPE-2 study is registered in the register of clinicaltrials.gov, Identifier: NCT01511276 .