ObjectivesProlonged sitting is associated with an increased risk of musculoskeletal pain, especially in nightshift workers. However, research investigating effects of breaking up sitting on musculoskeletal pain during nightshifts is lacking. This study evaluated effects of prolonged sitting or breaking up sitting with short bouts of light-intensity physical activity on pain in healthy adults during simulated nightshifts. MethodsAn in-laboratory randomised controlled trial was undertaken with 52 healthy adults completing five simulated nightshifts. Participants were randomised to prolonged sitting (Sit9; n = 26) or breaking up prolonged sitting (Break9; n = 26). Break9 group completed 3-min walking every 30 min during nightshifts, while Sit9 group remained seated. Musculoskeletal pain intensity and sensory/affective pain experiences were assessed. Linear mixed models examined pain within nights (pre-to post-shift) and across nights (pre-shift-night-1 to pre-shift-night-5). ResultsMusculoskeletal pain intensity increased within nights for both Sit9 (mean change [95%CI] points: 0.14 [0.05, 0.24]) and Break9 (0.09 [0.001, 0.19], but not across nights (Sit9: −0.13 [-0.33, 0.08]; Break9: 0.07 [-0.14, 0.29]). Sensory-pain experience improved across nights for Sit9 (−3.08 [-4.72, −1.45]), but not within nights (0.77 [-0.004, 1.55]). There was no change in affective-pain experience in either group. Between-group difference was observed favouring Sit9 for improving sensory-pain across nights (β: 3.71 [1.42, 5.99]). No other between-group difference was observed. ConclusionBoth prolonged sitting and breaking up sitting were associated with a within-night increase in musculoskeletal pain intensity. Compared to prolonged sitting, breaking up sitting did not induce benefits on pain in healthy adults working simulated nightshifts. Trial registrationACTRN12619001516178.