Objectives: To investigate whether health-related physical fitness is associated with total and central body fat in preschool children. Methods: A total of 403 Spanish children aged 3-5 years (57.8% boys) participated in the study. Health-related physical fitness was measured by the PREFIT battery: the handgrip strength and the standing long-jump tests (muscular strength), the 4 x 10 m shuttle run (speed-agility), the one-leg stance tests (balance) and the PREFIT-20 m shuttle run test (cardiorespiratory fitness). Body mass index and waist circumference were used as markers of total and central body fat, respectively. Results: There were significant associations between all health-related physical fitness tests and body mass index (beta = 0.280 +/- 0.054, beta = -0.020 +/- 0.006, beta = 0.154 +/- 0.065 and beta = -0.034 +/- 0.011 for the handgrip strength, standing long jump, 4 x 10 m shuttle run and PREFIT-20 m shuttle run tests, respectively, all P <= 0.019) after adjusting for sex and age. Similarly, there was significant associations of standing long jump (beta = -0.072 +/- 0.014), 4 x 10 m shuttle run (beta = 0.652 +/- 0.150) and PREFIT-20 m shuttle run tests (beta = -0.102 +/- 0.025) with waist circumference (all P <= 0.001), except for handgrip strength (beta = 0.254 +/- 0.145, P=0.081) and one-leg stance (beta = -0.012 +/- 0.009, P=0.156). Conclusions: The present study extends previous findings in older youth. Fitness assessment should be introduced in future epidemiological and intervention studies in preschool children because it seems to be an important factor determining health.