Objectives This study aimed to investigate arterial stiffness index, physical activity, and food and nutrient intake in middle-aged adults over 40 years when the incidence of cardiovascular disease begins to increase. Methods This study included 106 subjects (48 males and 58 females) aged between 40 and 64 years. The arterial stiffness index (brachial-ankle pulse wave velocity [baPWV], and anklebrachial index [ABI]) were measured using a blood pressure pulse wave testing device. Physical activity was assessed using the Korean version of the Global Physical Activity Questionnaire, and food and nutrient intake was calculated using the Food Frequency Questionnaire. Results The mean age of the subjects was 54.4 years. Although the ABI of the subjects was within the normal range, they were divided into tertiles to compare physical activity and food and nutrient intake. In males, the time spent on moderate to vigorous physical activity (MVPA) was significantly higher in T3 (600.6 min/week) than in T1 (304.4 min/week). In females, the time spent in sedentary behavior was significantly lower in T3 (294.5 min/week) than in T1 (472.1 min/week). In addition, the frequency of fish consumption was significantly higher in T3 (1.27 frequency/day) than in T1 (0.64 frequency/day) in females. Polyunsaturated fatty acid (PUFA) and ω-3 fatty acid intake, adjusted for energy intake, were significantly positively correlated with ABI (r = 0.200 and r = 0.218, respectively). Conclusions High MVPA (in males), low sedentary behavior (in females), and PUFA and ω-3 fatty acid intake through fish consumption may be associated with low peripheral artery stiffness. Therefore, arteriosclerosis can be prevented through physical activity and proper dietary therapy.