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Physically active women have a reduced risk of breast cancer, but the dose of activity necessary and the role of energy balance and other potential mechanisms have not been fully explored in animal models. We examined treadmill and wheel running effects on mammary tumorigenesis and biomarkers in p53-deficient (p53+/−):MMTV-Wnt-1 transgenic mice.Female mice (9 wk old) were randomly assigned to the following groups in experiment 1: treadmill exercise 5 d·wk−1, 45 min·d−1, 5% grade at 20 m·min−1, ∼0.90 km·d−1 (TREX1, n = 20) or at 24 m·min−1, ∼1.08 km·d−1 (TREX2, n = 21); or a nonexercise control (CON-TREX, n = 22). In experiment 2, mice were randomly assigned to voluntary wheel running (WHL, n = 21, 2.46 ± 1.11 km·d−1 (mean ± SD)) or to a nonexercise control (CON-WHL, n = 22). Body composition was measured at ∼9 wk and serum insulin-like growth factor 1 (IGF-1) at two to three monthly time points beginning at∼9 wk on study. Mice were sacrificed when tumors reached 1.5 cm, mice became moribund, or there was only one mouse per treatment group remaining.TREX1 (24 wk) and TREX2 (21 wk) had shorter median survival times than CON-TREX (34 wk; P < 0.01), whereas those of WHL and CON-WHL were similar (23 vs 24 wk; P = 0.32). TREX2 had increased multiplicity of mammary gland carcinomas compared with CON-TREX; WHL had a higher tumor incidence than CON-WHL. All exercising animals were lighter than their respective controls, and WHL had lower body fat than CON-WHL (P < 0.01). There was no difference in IGF-1 between groups (P > 0.05).Despite beneficial or no effects on body weight, body fat, or IGF-1, exercise had detrimental effects on tumorigenesis in this p53-deficient mouse model of spontaneous mammary cancer.