Muscle activation during interruptions to prolonged sedentary time is a hypothesized mechanism underlying observed cardiometabolic benefits. We examined associations of quadriceps and hamstring muscle activity patterns with cardiometabolic risk markers and how these patterns varied between different sitting-interruption countermeasures. Electromyographic (EMG) data (shorts) were gathered for 1 to 2 days from healthy adults in a free-living study (n = 172, age 40.9 ± 12.9, BMI 23.6 ± 1.3) and a laboratory-based study (n = 12, age 47.0 ± 7.7, BMI 30.0 ± 4.7). Patterns examined were average EMG (aEMG ;%EMGMVC); EMG activity duration (% above signal baseline 3 μV); and usual (weighted medians) EMG activity bout amplitude (%EMGMVC) and duration (s). In the free-living study, these were regressed against risk markers (waist; fat percentage; fasting plasma glucose [FPG];total cholesterol; HDL;LDL; triglycerides); in the laboratory study, EMG patterns for the muscle groups were compared between sitting and the active countermeasures. In the free-living study, lower extremity muscles displayed minimal overall activity, with hamstrings and quadriceps using only 2.6% and 2.0% of their capacity (%EMGMVC), respectively, and being active for 30% and 25% of the time. Higher hamstring aEMG and EMG activity duration were beneficially associated with waist, HDL and fat percentage (duration only) and a longer quadriceps usual EMG activity bout duration was beneficially associated with FPG. In the laboratory study, compared with prolonged sitting, active seated or upright active-interruption countermeasures modified these EMG patterns; brief (6 min) walking and simple resistance activities (SRA) were more beneficial than was a bout of standing (30 min) with the SRAs being the only intervention that matched daily aEMG levels. Upright and physically active interruptions to sitting appear to be required to increase the typically low muscle engagement observed in free-living contexts, promoting muscle activity patterns that may help ameliorate cardiometabolic risk.