High physical activity levels during wake are beneficial for health, while high movement levels during sleep are detrimental to health. Our aim was to compare the associations of accelerometer-assessed physical activity and sleep disruption with adiposity and fitness using standardized and individualized wake and sleep windows. People (N = 609) with type 2 diabetes wore an accelerometer for up to 8 days. Waist circumference, body fat percentage, Short Physical Performance Battery (SPPB) test score, sit-to-stands, and resting heart rate were assessed. Physical activity was assessed via the average acceleration and intensity distribution (intensity gradient) over standardized (most active 16 continuous hours (M16h)) and individualized wake windows. Sleep disruption was assessed via the average acceleration over standardized (least active 8 continuous hours (L8h)) and individualized sleep windows. Average acceleration and intensity distribution during the wake window were beneficially associated with adiposity and fitness, while average acceleration during the sleep window was detrimentally associated with adiposity and fitness. Point estimates for the associations were slightly stronger for the standardized than for individualized wake/sleep windows. In conclusion, standardized wake and sleep windows may have stronger associations with health due to capturing variations in sleep durations across individuals, while individualized windows represent a purer measure of wake/sleep behaviors.