Prolonged sedentary behaviour has a detrimental effect on neuromuscular function and is associated with decreased muscle strength and force control, and a decreased ability to maintain balance. Breaking up sedentary time with regular bouts of physical activity has numerous health benefits, though the effects on neuromuscular function are unknown. This study aimed to investigate the effect of breaking up sedentary time with calisthenic exercise on neuromuscular function. To that end, 17 healthy adults, who spent ≥6 hours a day sitting, were randomly assigned to a four-week calisthenics intervention (n = 8) or a control group (n = 9). The calisthenics intervention involved performing up to eight sets of exercises during the working day (09:00-17:00); with one set consisting of eight repetitions of five different exercises (including squats and lunges). Before and immediately after the intervention, measures of knee extensor maximal voluntary contraction (MVC; right leg only) and submaximal force control (measures of the magnitude and complexity of force fluctuations; right leg only), and dynamic balance (Y balance test; both legs) were taken. The calisthenics intervention resulted in a significant increase in knee extensor MVC (P = 0.036), significant decreases in the standard deviation (P 0.031) and coefficient of variation (P = 0.016) of knee extensor force fluctuations during contractions at 40% MVC, and a significant increase in Y balance test posterolateral reach with left leg stance (P = 0.046). These results suggest that breaking up sedentary time with calisthenics may be effective at increasing muscle strength, force steadiness and dynamic balance.