The purpose of this study was to investigate photothermal aspects of photobiomodulation therapies (PBMT) in vitro to assist in the development of safe clinical parameters with respect to higher-power devices with large surface applicators. Laser wavelengths in the range of 650 nm–1064 nm were investigated using a thermal camera. Thermographic measures of surface and sub-surface temperature variations of similar lean porcine muscle tissue samples were recorded for a series of calibrated experiments. A thermal comparison was then made between Flat-top and Gaussian beam spatial distribution devices. Outcome data were subjected to statistical analysis using an ANOVA model. Results acquired at similar parameters of irradiance indicated that the application of the 980 nm wavelength was associated with the highest rise in temperature, which decreased with other wavelengths in the order 980 > 1064 ≈ 650 >>> 810 nm (p< 5 × 10−20). All wavelengths assessed were associated with a significant temperature increase, and with the exception of 810 nm, all exceeded the threshold of a 6 °C rise within the prescribed parameter limits. Optical scanning by movement of the applied source over a relevant area was found to offer effective mitigation of these temperature increases. An extended discussion is presented, analysing the clinical significance of the study outcomes. Recommendations are made within the limits of this in vitro study in order to assist future clinical investigations.