Acute Effects of Interrupting Prolonged Sitting on Glucose, Insulin and Lipid Metabolism in Healthy Populations: A Three-Level Meta-Analysis

Background/Objectives: Prolonged sedentary behavior is associated with adverse cardiometabolic risk, but the acute metabolic effects of interrupting sitting remain incompletely quantified in apparently healthy populations. The objective was to evaluate the acute effects of interrupting prolonged sitting on glucose, insulin and lipid metabolism and to identify potential protocol-level moderators. Methods: We conducted a three-level meta-analysis of randomized crossover trials comparing structured sitting interruptions with uninterrupted sitting in apparently healthy participants, an approach used to account for the statistical dependency among multiple effect sizes reported within individual studies. PubMed, Embase, Web of Science, Scopus and Cochrane Library were searched from inception through March 2026. Standardized mean differences (Hedges’ g) were calculated using random-effects models with robust variance estimation. Subgroup analyses and meta-regression examined interruption modality, intensity, frequency and duration. Certainty of evidence was assessed using GRADE. Results: The evidence base comprised 25 publications representing 23 independent cohorts; primary outcome analyses included 23 cohorts (135 effect sizes). Sitting interruption produced small but significant reductions in glucose (g = −0.28, 95% CI −0.49 to −0.08; PI −1.15 to 0.59) and insulin (g = −0.22, 95% CI −0.36 to −0.07; PI −0.51 to 0.07). No significant effect was observed for triglycerides (g = −0.04, 95% CI −0.14 to 0.07), whereas the exploratory estimates for non-esterified fatty acids (NEFA) and C-peptide were inconclusive. Subgroup analyses did not identify any statistically significant moderators of glucose, insulin or triglyceride responses; all subgroup estimates drawn from fewer than five independent cohorts are exploratory and hypothesis-generating. Meta-regression identified no statistically significant linear moderators. Certainty of evidence was low for all three primary outcomes (glucose, insulin and triglycerides) and very low for the exploratory outcomes (NEFA and C-peptide). Conclusions: Interrupting prolonged sitting may produce modest acute improvements in glucose and insulin responses in apparently healthy participants but does not reliably affect triglyceride metabolism. Given the low certainty of the current evidence, sitting interruption may be considered a low-burden adjunctive behavior for reducing uninterrupted sitting time. However, the current evidence primarily supports the hypothesis that interrupting prolonged sitting is a promising behavioral strategy rather than an evidence-based clinical recommendation, and its metabolic effects require confirmation in adequately powered, higher-quality trials.