Although sleep quality and physical activity (PA) may influence on arterial stiffness, the combined effects of these two factors on arterial stiffness remain unknown. A total of 103 healthy middle-aged and older men and women (aged 50–83 years) with no history of cardiovascular disease and depression were included in this study. Arterial stiffness was measured using carotid-femoral pulse wave velocity (cfPWV), brachial-ankle PWV (baPWV), and femoral-ankle PWV (faPWV). Poor sleepers were defined as those with a Pittsburgh Sleep Quality Index score of >5.5. Using an accelerometer for seven consecutive days, low levels of PA were defined as low moderate-to-vigorous-intensity PA (MVPA) <19.0 min/day and low step counts <7100 steps/day, respectively. Poor sleepers with low PA levels, as determined by MVPA and daily steps, showed higher cfPWV, but not faPWV or baPWV, in middle-aged and older adults. Furthermore, in the analysis of covariance (ANCOVA) analyses adjusted for age, obesity, dyslipidemia, and sedentary behavior, the cfPWV result remained significant. Our study revealed that the coexistence of poor sleep quality and decreased PA (low MVPA or daily steps) might increase central arterial stiffness in middle-aged and older adults. Therefore, adequate sleep (good and sufficient sleep quality) and regular PA, especially at appropriate levels of MVPA (i.e., at least of 7100 steps/day), should be encouraged to decrease central arterial stiffness in middle-aged and older adults. [ABSTRACT FROM AUTHOR]