Evidence on whether habitual sleep duration and sleep quality are associated with increased insulin resistance is inconsistent. Here, we investigated the associations between different measures of habitual sleep with glycemic traits through cross-sectional and Mendelian randomization (MR) analyses. We assessed the associations of sleep duration and sleep quality with glycemic traits using multivariable linear regression models adjusted for potential confounders in 4672 middle-aged (45–65 years; 48% men) nondiabetic participants of the Netherlands Epidemiology of Obesity (NEO) study. Genetic variants for total, short, and long sleep duration were used as instrumental variables in MR analyses using summary-level data of glycemic traits in nondiabetic individuals (MAGIC; n = 58,074). In cross-sectional analyses, shortest sleepers (median 5.0 h of sleep per night) had 14.5% (95% confidence interval (CI): 2.0; 28.6%) higher fasting insulin level and 16.3% (95% CI: 2.7; 31.7%) higher HOMA-β. Bad sleep quality was associated with higher insulin resistance (e.g., 14.3% (95% CI: 4.7; 24.9%) higher HOMA-IR). All these associations disappeared after adjustment for BMI and the risk of sleep apnea. MR analyses did not indicate a causal association between total, short or long sleep duration and glycemic traits. Therefore, our used measures of habitual sleep duration and sleep quality are unlikely to directly associate with insulin resistance.