Breast cancer (BC) is the most common cancer in the UK and almost 25% of BC’s present with an aggressive form characterised by increased expression of human epidermal growth receptor 2 (HER2) proteins. In these patients, trastuzumab (TRZ) can be used to inhibit HER2 signalling pathways resulting in reduced tumour proliferation and improved survival. Unfortunately, TRZ can also cause cardiotoxicity which, via impairment of HER2 cardioprotective pathways, can result in congestive heart failure. At present, the precise mechanisms for this are unclear, although endothelial dysfunction has been hypothesised as a possible cause. Thus, the present study examined microvessel and large vessel endothelial function, arterial stiffness and carotid intima-media thickness (cIMT) in female breast cancer patients and healthy controls. Thirteen TRZ-naïve BC patients (age: 57 ± 15 years) with HER2 positive tumours who had been treated with TRZ for at least one year, as well as 15 healthy controls (age: 54 ± 9 years) underwent assessments of microvascular endothelial function (laser Doppler imaging with iontophoresis of endothelium-dependent agonist – acetylcholine (ACh) and endothelium-independent agonist – sodium nitroprusside), large vessel endothelial function (flow-mediated dilatation (FMD) and glyceryl-trinitrate mediated dilatation), arterial stiffness (pulse wave analysis), and cIMT. Cardiovascular disease risk was also calculated in both groups using the Framingham Risk Score (FRS) and QRISK2. Univariate Analysis of Variance revealed no significant differences in age, systolic or diastolic blood pressure, FRS or QRISK2 between groups (p’s > 0.3). However, TRZ patients had greater body mass index (BMI) and body fat percentage (Fat %) relative to healthy controls (BMI: 29 ± 6 and 26 ± 3kg/m2 respectively, p = 0.04; Fat%: 37 ± 9 and 31 ± 5% respectively, p = 0.03). TRZ-treated patients also had significantly worse microvascular perfusion in response to ACh when compared with healthy controls (TRZ: 267 ± 130% vs. healthy controls: 545 ± 355%, p = 0.01), along with worse large vessel endothelial function (FMD: 9.9 ± 7.8 and 16 ± 7 % respectively, p = 0.04). Univariate Analysis of Covariance revealed that the difference in microvascular endothelium-dependent function between groups remained significant even after adjustment for BMI and fat % (p = 0.03), but large vessel endothelial function was no longer significantly different. No significant differences were found for arterial stiffness or cIMT. The present findings reveal that in BC patients treated with TRZ, there is evidence of functional but not structural impairments in the vasculature, and that these changes are confined mainly to the microvessels. Further prospective studies examining the time-course of endothelial dysfunction following TRZ treatment are warranted.