Background Skin microvascular responses to local heating are frequently used to assess microvascular function. Several local heating protocols have been developed, all varying slightly in execution. The aim of this study was to determine the inter-day reproducibility of the four most commonly used local heating protocols in healthy young subjects. Methods Fifteen, healthy males (28 ± 5 yrs, BMI 25 ± 2 kg/m2) attended two experimental trials 2–7 days apart. During each trial, baseline and maximal thermally stimulated forearm skin responses were examined simultaneously at four sites on the dominant forearm using laser Doppler flowmetry (LDF). The following heating protocols were adopted: 1. Rapid 39 °C (0.5 °C/5 s), 2. Rapid 42 °C (0.5 °C/5 s) 3. Gradual 42 °C (0.5 °C/2 min 30 s) and 4. Slow 42 °C (0.5 °C/5 min). The coefficient of variation (CV) was calculated for absolute flux, cutaneous vascular conductance (CVC; flux/mean arterial pressure, MAP) and CVC expressed as a percentage of maximal CVC at 44 °C (%CVCmax) at three different time points; baseline (33 °C), plateau (39/42 °C) and maximal (44 °C). Results Reproducibility of baseline flux, CVC and %CVCmax was 17–29% across all protocols. During the plateau, Rapid, Gradual and Slow 42 °C demonstrated a reproducibility of 13–18% for flux and CVC and 5–11% for %CVCmax. However, Rapid 39 °C demonstrated a lower reproducibility for flux, CVC and %CVCmax (all 21%). Reproducibility at 44 °C was 12–15% for flux and CVC across all protocols. Conclusion This is the first study examining inter-day reproducibility across four local heating protocols. The good-to-moderate reproducibility of the Rapid, Gradual and Slow 42 °C protocols support their (simultaneous) use to assess microvascular function. Using Rapid 39 °C may require a greater number of subjects to detect differences within subjects.