© 2022 The AuthorsBackground: Women treated for breast cancer are at risk for worsening health-related quality of life (QoL), cardiac function, and cardiorespiratory fitness. Objectives: The aim of this study was to assess the associations of self-reported moderate to vigorous intensity physical activity (MVPA) during cancer treatment with concurrent measures of QoL and cardiac function and with post-treatment cardiorespiratory fitness in women with human epidermal growth factor receptor 2–positive breast cancer receiving sequential anthracyclines and trastuzumab. Methods: EMBRACE-MRI 1 (Evaluation of Myocardial Changes During Breast Adenocarcinoma Therapy to Detect Cardiotoxicity Earlier With MRI) study participants who completed questionnaires for MVPA (modified Godin Leisure Time Physical Activity Questionnaire) and QoL (EQ-5D-3L, Minnesota Living With Heart Failure Questionnaire) and cardiac imaging every 3 months during treatment and post-treatment cardiopulmonary exercise testing were included. Participants engaging in ≥90 minutes of MVPA each week were labeled “active.” Generalized estimation equations and linear regression analyses were used to assess concurrent and post-treatment associations with MVPA and activity status, respectively. Results: Eighty-eight participants were included (mean age 51.4 ± 8.9 years). Mean MVPA minutes, QoL, and cardiac function (left ventricular ejection fraction, global longitudinal strain, E/A ratio, and E/e′ ratio) worsened by 6 months into trastuzumab therapy. Higher MVPA (per 30 minutes) during treatment was associated with better concurrent overall (β = −0.42) and physical (β = −0.24) Minnesota Living With Heart Failure Questionnaire scores, EQ-5D-3L index (β = 0.003), visual analogue scale score (β = 0.43), diastolic function (E/A ratio; β = 0.01), and global longitudinal strain (β = 0.04) at each time point (P ≤ 0.01 for all). Greater cumulative MVPA over the treatment period was associated with higher post-treatment cardiorespiratory fitness (peak oxygen consumption; β = 0.06 per 30 minutes; P < 0.001). Conclusions: Higher self-reported MVPA during treatment for human epidermal growth factor receptor 2–positive breast cancer was associated with better QoL and diastolic and systolic left ventricular function measures during treatment and better post-treatment cardiorespiratory fitness.