Replacing sitting by standing has been hypothesized to reduce the health risks of sitting, based on the assumption that muscles are passive during sitting and active during standing. Interventions have been more effective in overweight (OW) than in normal weight (NW) individuals, but subjects’ muscle activities have not been quantified. This study compared quadriceps and hamstring muscle electromyographic (EMG) activity between 57 NW (body mass index (BMI) 22.5 ± 1.5 kg/m2, female n = 36) and 27 OW (BMI 28.4 ± 2.9 kg/m2, female n = 8) subjects during non-fatiguing standing (15 s, EMGstanding) and sitting (30 min). EMG amplitude was normalized to EMG measured during maximal isometric knee extension and flexion (% EMGMVC), and sitting muscle inactivity and bursts were determined using 4 thresholds (60% or 90% EMGstanding and 1% or 2% EMGMVC). Comparisons were adjusted for sex, age, knee extension strength, and the individual threshold. Standing EMG amplitude was 36% higher in OW (1.9% ± 1.5% EMGMVC) than in NW (1.4% ± 1.4% EMGMVC, P < 0.05) subjects. During sitting, muscles were inactive 89.8% ± 12.7% of the measurement time with 12.7 ± 14.2 bursts/min across all thresholds. On average, 6% more activity was recorded in NW than in OW individuals for 3 of the 4 thresholds (P < 0.05 for 60% or 90% EMGstanding and 1% EMGMVC). In conclusion, the OW group had higher muscle activity amplitude during standing but more muscle inactivity during sitting for 3/4 of the thresholds tested. Interventions should test whether the observed heterogeneity in muscle activity affects the potential to gain cardiometabolic benefits from replacing sitting with standing.