Accelerometry is a commonly used method to determine physical activity in clinical studies, but the duration and timing of measurement have seldom been addressed. We aimed to evaluate possible changes in the measured outcomes and associations with insulin resistance during four weeks of accelerometry data collection. This study included 143 participants (median age of 59 (IQR9) years; mean BMI of 30.7 (SD4) kg/m²; 41 men). Sedentary and standing time, breaks in sedentary time, and different intensities of physical activity were measured with hip-worn accelerometers. Differences in the accelerometer-based results between weeks 1, 2, 3 and 4 were analyzed by mixed models, differences during winter and summer by two-way ANOVA, and the associations between insulin resistance and cumulative means of accelerometer results during weeks 1 to 4 by linear models. Mean accelerometry duration was 24 (SD3) days. Sedentary time decreased after three weeks of measurement. More physical activity was measured during summer compared to winter. The associations between insulin resistance and sedentary behavior and light physical activity were non-significant after the first week of measurement, but the associations turned significant in two to three weeks. If the purpose of data collection is to reveal associations between accelerometer-measured outcomes and tenuous health outcomes, such as insulin sensitivity, data collection for at least three weeks may be needed © 2021 by the author. Licensee MDPI, Basel, Switzerland.