Background: The number of cancer survivors across North America is increasing, with estimates indicating that this population will grow to nearly 19 million individuals by the end of 2020. This increase can be attributed, in part, due to improvements in cancer treatments, of which radiation therapy is most commonly used. Unfortunately, exposure to radiation also increases the risk of secondary cancer development long-term. Moreover, obesity and physical inactivity are prevalent, modifiable, risk factors among cancer survivors, with both factors being linked to decrements in quality of life, increased cancer risk, and greater mortality risk. To date, there has been promising epidemiological and clinical data highlighting the role of exercise as a way to mitigate cancer risk and improve survival; however, longitudinal studies are lacking and the effects of radiation in these studies have been largely ignored. Therefore, there is a major clinical need to directly evaluate the combinatory long-term effects of radiation, exercise, and/or obesity to reveal their implications on healthspan, cancer incidence, and survival. Recent pre-clinical work from our group has shown that after being exposed to radiation, endurance exercise prevented several negative alterations to hematopoietic stem cells and their niche caused by high-fat diet (HFD)-induced obesity. We also showed that leukemic blast viability in vitro was greater when cultured in bone marrow supernatant from mice with HFD-induced obesity compared to bone marrow supernatant from mice without HFD-induced obesity. It is unknown, however, whether these findings extend to alterations in cancer risk across the lifespan. As such, the purpose of this study was to evaluate the effects of lifelong exercise and diet-induced obesity on healthspan, cancer incidence, and survival in an established mouse model of radiation-induced cancer. Methods: Male CBA mice (n=80) were randomly divided into either a control diet (CTRL; n=40) of 45% high-fat diet (HFD; n=40) and then further divided into either a sedentary group (SED; n=20) or exercise-trained group (EX; n=20). At age 13 weeks, all mice were exposed to a cancer inducing dose of whole-body ionizing radiation (3 Gy). A healthspan index score and endpoint monitoring were conducted throughout the study by blinded investigators. Results: When normalized to CTRL/SED, the highest healthspan score was in the CTRL/EX (score = +2.5), followed by HFD/EX (score = +1) and lastly HFD/SED (score = -0.5). Cancer incidence was significantly higher in the HFD/SED group when compared to the CTRL/EX group (p<0.05) and a trend for higher cancer incidence for HFD/SED was observed when compared to the CTRL/SED group (p=0.079). There was no significant difference between the HFD/SED and HFD/EX group in cancer incidence (p>0.05). Overall survival was significantly higher in the HFD/SED group compared to CTRL/SED group (p<0.05); however, risk of cancer-related mortality was 1.6-times higher in the HFD/SED group compared to the CTRL/SED group (RR=1.60; 95% CI, 1.00-2.56; p=0.0495) and 1.68-times that of the CTRL/EX group (RR=1.68; 95% CI, 1.02-2.78; p=0.0415). Conclusion: Our findings show that lifelong exercise training resulted in higher healthspan index, lower cancer incidence, and lower risk of cancer-related mortality following radiation exposure, with these effects being largely reversed by HFD-induced obesity. This study provides the rationale for future studies to uncover cellular and molecular mechanisms that could be underlying these results. Moreover, this study presents a proof of concept for the consideration of clinical studies in cancer survivors examining exercise as an intervention to reduce the long-term effects of radiation.