Background <p>Physical activity is a critical determinant of health in aging populations. Although the health benefits of exercise are well established, limited research has examined how physical activity patterns and rest-activity rhythms influence biological rhythms and cognitive performance. This study aimed to investigate the relationships between physical activity, rest-activity rhythm, melatonin, and sustained attention in older adults.</p> Methods <p>We recruited 147 community-dwelling adults aged ≥ 60 years, who were instructed to wear wrist accelerometers for 14 consecutive days. From the accelerometry data, we derived measures of daytime physical activity (most active 10&#xa0;h), the distribution of moderate-to-vigorous activity, and 24&#xa0;h rest-activity rhythm parameters, including interdaily stability, amplitude, autocorrelation coefficient, and relative amplitude. Salivary melatonin was collected at home, and dim-light melatonin onset was calculated. Sustained attention was assessed using a 3-minute smartphone-based psychomotor vigilance task.</p> Results <p>Greater amounts of daytime physical activity and a higher proportion of high-intensity activity were associated with more robust rest-activity rhythms, as evidenced by higher interdaily stability, relative amplitude, 24&#xa0;h autocorrelation, and activity amplitude. In turn, higher interdaily stability was associated with greater peak melatonin levels (β = 39.38; 95% CI = 6.02 to 72.73). In the psychomotor vigilance task, higher relative amplitude was associated with fewer lapses (β = -32.61; 95% CI = -55.60 to -9.62).</p> Conclusions <p>Physical activity is associated with a stronger rest-activity rhythm, which further associated with and better health outcomes in older adults. Future studies are warranted to clarify the interactions between physical activity, rest-activity patterns, and biological rhythms.</p>