Objectives: First, to determine the association between serum 25 hydroxyvitamin D (25OHD) concentration and muscle mass, strength, and performance. Second, to explore if there is a threshold in the association. Design: Cross-sectional, single-center study. Setting: The central part of the Netherlands (52 degrees Northern latitude). Participants: A total of 802 independently living men and postmenopausal women 40 to 80 years of age. Measurements: Health-related and lifestyle factors, including physical activity, 25OHD concentration, lean mass, handgrip strength, knee extension strength, and physical performance were determined. Results: Overall, higher 25OHD level was significantly associated with higher lean mass (22.6 g per nmol/L, 95% CI 7.3-37.9), handgrip strength (0.020 kg per nmol/L, 95% CI 0.001-0.038), and physical performance (0.006 points per nmol/L, 95% CI 0.001-0.012), after adjustment for various confounders. This association was most pronounced below a 25OHD level of 60 nmol/L, with lean mass increase 79.6 g per nmol/L (95% CI 40.8-118.4, P < .01), handgrip strength 0.09 kg per nmol/L (95% CI 0.045-0.141, P < .01), and physical performance 0.02 points per nmol/L (95% CI 0.005-0.032, P < .01), and these significant associations attenuated to null above this threshold. Conclusion: In middle-aged men and (postmenopausal) women, a higher 25OHD level was significantly associated with higher lean mass, muscle strength, and performance. These associations were most pronounced below 60 nmol/L and absent above 60 nmol/L, indicating a ceiling effect. Copyright (C) 2013 - American Medical Directors Association, Inc.