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While the lack of muscular activity is a proposed trigger for metabolic alterations, this association has not been directly measured. We examined the associations between EMG-derived muscle inactivity and activity patterns and cardiometabolic biomarkers in healthy, physically active adults.Data for this cross-sectional study were pooled from two studies (EMG24 and InPact), resulting in a sample of 150 individuals without known chronic diseases and with high-quality EMG data (female n = 85, male n = 65, age = 38.8 ± 10.6 yr, body mass index = 23.8 ± 3.1 kg·m−2). EMG was measured during one to three typical weekdays using EMG shorts, measuring quadriceps and hamstring muscle EMG. Muscle inactivity time and moderate- to vigorous-intensity muscle activity were defined as EMG amplitude below that of standing still and above that of walking 5 km·h−1, respectively. Blood pressure index, waist circumference, fasting plasma glucose, HDL cholesterol, and triglycerides were measured, and long-term exercise behaviors were assessed by questionnaire.In a group of physically active participants, muscles were inactive for 65.2% ± 12.9% of the measurement time in an average of 24.1 ± 9.8-s periods. Compared to those in the lowest muscle inactivity quartile (<55.5% of measurement time), those in the highest quartile (≥74.8% of measurement time) had 0.32 mmol·L−1 lower HDL cholesterol (P < 0.05) and 0.30 mM higher triglycerides (P < 0.05) independent of muscle’s moderate- to vigorous-intensity activity.Clinically significant differences in HDL cholesterol and triglycerides were found, favoring participants having low muscle inactivity time, independent of moderate- to vigorous-intensity muscle activity. Even physically active individuals may benefit from light-intensity activities that reduce ubiquitous muscle inactivity time.