Objective Endoscopic laser therapy has been proposed as a useful tool for inducing coagulative tissue necrosis in the treatment of varicosis, vascular lesions as well as benign and malignant tumors. This method often relies on near-infrared or infrared wavelengths, where hemoglobin and water are assumed to be the main chromophores, and light sources providing output powers in the 1–100 W range. For an accurate prediction of the treatment volume, a profound knowledge of the interaction of light with tissue is required. Here the impact of 980-nm- and 1470-nm-light application in whole blood was investigated by performing time-resolved temperature measurements. The experimental observations were compared to theoretical simulations of the light and temperature distributions. Material and methods A 5-ml glass vial with a diameter exceeding the optical penetration depth at the two wavelengths investigated was filled with bovine blood. An optical fiber with a cylindrical diffusing tip (1.6 mm diameter, 2 mm length), a light source typically used for endovenous laser therapy (ELT), was placed centrically in the vial. The temperature was monitored via an optical fiber, which was equipped with a ruby-doped tip and positioned parallel to the cylindrical diffuser. Via temperature-dependent variations of the fluorescence spectrum of the ruby crystal, the temperature can be measured in the range 30–200 °C with an accuracy of ±1 °C. Hyperthermia was induced in the blood by applying laser light at 980 and 1470 nm at 10 W for 15–120 s. Simulations corresponding to the experimental set-up were performed with MATLAB's Partial Differential Equation toolbox (The Mathworks™). The light distribution was modeled on the diffusion equation for light transport. Wavelength-dependent light absorption was assumed due to hemoglobin and water. The light-induced temperature increase was modeled by the heat transfer equation including heat conduction and convection. However, it did not take into account the possibility of inducing blood coagulation. Results Experiments resulted in rapid temperature increases (up to 160 °C) and coagulation of blood following application of both wavelengths. The light-induced zones of coagulation (diameters up to 10 mm) were centered up the cylindrical diffuser. A strong light attenuation was observed within the volume of blood coagulation, preventing further light penetration. Application of equal light doses resulted in significantly higher temperatures for 980 nm as compared to 1470 nm. In contrast to the experimental results, the simulations indicated a higher temperature increase for the longer wavelength. The discrepancies between experimental results and simulations are likely due to the assumption of blood as a spatially homogenous absorber. The experimentally observed zones of coagulation cause a significant change of the absorption, scattering, and heat transfer coefficients. These changes further increase light absorption close to the light source, leading to rapidly increasing temperatures. Conclusion The fundamental differences in light distribution and temperature increase following interstitial light delivery in bovine blood observed for experiments and simulations indicate the presence of tissue alterations during light application. These alterations, proposed here to consist of increased absorption and scattering coefficients, lead to temperature kinetics being different from what can be predicted by assuming light absorption by spatially homogeneous hemoglobin and water concentrations. Particularly the occurrence of blood coagulation close the light source needs to be taken into account when further developing hyperthermia as a tool for inducing selective tissue necrosis.