Perimenopause is the period of transition when midlife women experience increases in cardiovascular disease (CVD) risk. Arterial stiffness (AS) is a preclinical CVD risk factor that precedes the manifestation of clinical CVD. Hot flashes (HF) are bothersome symptoms of menopause which are correlated with some subclinical CVD risk factors, but the exact relationship between HF and CVD risk factors remain equivocal. Habitual physical activity (PA) is effective in the prevention and treatment of CVD in many populations. However, the relationship between PA and CVD risk remains unclear for perimenopausal women. Therefore, the objective of our study was to determine the effects of HF and PA on AS in healthy perimenopausal women. We hypothesized that HFs would be associated with AS and PA would influence the association between HFs and AS. There were 61 participants (age 43-54) free from CVD risk factors included in this analysis. Arterial stiffness was measured via augmentation index (AIx), backwards wave (PB, mmHg), and reflection magnitude (RM, %). Hot flashes were measured objectively for 24 hours using ambulatory sternal skin conductance monitoring. Objective HF rate was calculated by normalizing HF count to wear time. Habitual PA activity was quantified objectively as average daily minutes of waking moderate-vigorous PA (MVPA) over 7-day using actigraphy and the R package GGIR. All statistical analyses were performed using R (version 4.2.3) and RStudio (2023.06.2+561). Pearson’s correlation and multiple regression were used to evaluate the associations between dependent and outcome variables. Age and body mass index (BMI) were controlled, and assumption testing was completed for all models. Objective HF rate was significantly positively correlated with PB, r(53) = 0.38, p < 0.01, and RM, r(53) = 0.35, p <0.01. Objective MVPA was significantly negatively associated with AIx, r(61) = -0.45, p <0.001, PB, r(61) = -0.29, p <0.05, and RM, r(61) = -0.34, p < 0.01. Objective HF rate significantly predicted arterial stiffness variables, AIx (β = 14.58, p =0.05; adjR2 = 0.38), PB (β = 6.48, p<0.01; adjR2 = 0.24), and RM (β = 17.79, p <0.01; adjR2 = 0.39). Objective MVPA significantly predicted AIX (β = -0.12, p <0.01; adjR2 = 0.41), and RM (β = -0.06, p = 0.05; adjR2 = 0.29). When HF and MVPA were added in the same model, HF and MVPA were both significant predictors of RM (βHF = 16.25, p <0.01, βMVPA = -0.06, p <0.05; adjR2 = 0.42), MVPA only was significant with AIx (βHF = 11.38, p = 0.10, βMVPA = -0.13, p <0.01; adjR2 = 0.49), and HF only was significant with PB (βHF = 6.11, p <0.01, βMVPA = -0.02, p = 0.212; adjR2 = 0.25). Adding the interaction between HF and MVPA did not help explain variation in any AS variable. In conclusion, our data showed that more objectively-measured HF were associated with negative AS outcomes and that greater MVPA may be worth considering in decreasing arterial stiffness in perimenopausal women. Contrary to our hypothesis, MVPA did not help to explain the effects of HF on AS. Future studies should focus on understanding the mechanisms behind the negative relationship between hot flashes and arterial stiffness in midlife women. NIH NHLBI R15 Grant 1R15HL145650-01A1 (Witkowski); Smith College McKinley Honors Fellowship (Tha Ra Wun). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.