Purpose: To examine the acute effects of a reduced sitting day on executive function (EF) and musculoskeletal health in preschoolers. Methods: A sample of 29 children (54% boys; 4–5 y) participated in a randomized cross-over trial. Each child completed 2 protocols, which simulate a day at childcare in random order for 2.5 hours; a typical preschool day (50% sitting) and a reduced preschool day (25% sitting) where most sitting activities were replaced with standing activities. Sitting, standing, and stepping time were objectively assessed using an activPAL accelerometer. EF was evaluated using tablet-based EF assessments (inhibition, working memory, and task shifting). Musculoskeletal health was assessed using a handheld dynamometer and goniometer. Results: Compared with the typical preschool day, the reduced sitting day showed no significant differences for EF scores. Effect sizes for inhibition (d = 0.04), working memory (d = 0.02), and shifting (d = 0.11) were all small. For musculoskeletal health, no significant differences were reported after the reduced preschool day. The effect sizes for the hip extension force, hamstring flexibility, gastrocnemius length, and balancing on 1 leg were all small (d = 0.21, d = 0.25, d = 0.28, and d = 0.28). Conclusions: This study suggests that reducing sitting time is unlikely to result in acute changes in EF and musculoskeletal health among preschoolers.