Value of the Data •These data show that exercise-conditioned serum could be used in three-dimensional in vitro culture to evaluate the potential of exercise on cancer progression control. •The application of three-dimensional cell growth in soft agar offers the possibility to quantify cancer cell growth in response to exercise-conditioned sera avoiding in vivo models. •This model could be useful to compare the cancer progression control with different exercise protocols to personalize the prescription of exercise in terms of FITT principle (Frequency, Intensity, Time and Type). •Future experiments will be aimed to evaluate the effect of a single exercise bout during adjuvant treatment in breast cancer patients and in follow-up.