This study determined the effects of a physically inactive vs active lifestyle on high-risk (intra-abdominal (visceral), liver and intramuscular) fat in 16 (7 monozygotic and 9 dizygotic) middle-aged same-sex twin pairs with long-term discordance for physical activity habits. At baseline (1975), the active co-twins practiced more leisure-time activity than the inactive co-twins (p<0.0001), but they were no different in anthropometrics or work-related physical activity. Over the follow-up of 30 years, a significant difference in leisure-time activity persisted between the active and inactive co-twins and the inactive co-twins were less fit compared to their active co-twins. However, the most noticeable difference was in body fat distribution measured by magnetic resonance imaging. The physically inactive co-twins had 50% greater intra-abdominal fat area compared with the active co-twins. The liver fat score was 170% higher and the intramuscular fat area 54% higher among the inactive co-twins. This prospective study demonstrated that habitual physical activity could possibly prevent accumulation of high-risk fat such as intra-abdominal and ectopic adipose tissue, even after controlling for genetic liability. These results support the concept of preventing and targeting obesity by long-term maintenance of leisure-time physical activity.