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Abstract Background The effect of exercise on the microvasculature of patients with suspected coronary microvascular dysfunction (CMD), assessed by reactive hyperemia peripheral arterial tonometry (RH-PAT; EndoPAT), is unknown. The present study aimed to determine if standard clinical exercise stress testing (GXT) affected peripheral microvascular function, as determined by the reactive hyperemia index (RHI and LnRHI), in patients with suspected CMD. Methods In a cross-sectional study, patients (n = 76) were grouped based on whether the GXT was performed; 1) prior to (exercisers; n = 30), or 2) after the vascular assessment (non-exercisers; n = 46). Patients with an adenosine index of microvascular resistance > 25, adenosine coronary flow reserve (CFR) < 2.0, and/or acetylcholine CFR < 1.5 were considered to have CMD (n = 42). RHI and LnRHI quantified finger pulse amplitude hyperemia following 5 min of forearm ischemia. Results LnRHI was lower in patients with CMD compared to patients without CMD, while LnRHI was also lower in exercisers compared to non-exercisers (LnRHI: CMD Non-Exercisers: 0.63 ± 0.25; CMD Exercisers: 0.54 ± 0.19; No CMD Non-Exercisers: 0.85 ± 0.23; No CMD Exercisers: 0.63 ± 0.26; Condition and Exercise Main Effects: Both P < 0.01). In patients who did not exercise prior to the vascular assessment, the receiver operating characteristic curve (ROC) for LnRHI to predict CMD was 0.76 (95% CI: 0.62–0.91; P < 0.01). However, in patients who performed exercise prior to the vascular assessment, the ROC for LnRHI to predict CMD was 0.60 (95% CI: 0.40–0.81; P = 0.34). Conclusions CMD is associated with impaired peripheral microvascular function and preceding acute exercise is associated with further reductions of LnRHI. Further, acute exercise abolished the capacity for RH-PAT to predict the presence of CMD in patients with chest pain and non-obstructive coronary arteries. RH-PAT measurements in patients with suspected CMD should not be conducted after exercise has been performed.