Increasing numbers in interdisciplinary applications of Fiber-optic Distributed Temperature Sensing (FO-DTS) call for a quantitative assessment of the limitations and uncertainties of this new technology. This study conducts controlled laboratory experiments to analyze the qualitative (signal size and location) and quantitative (signal intensity) accuracies of FO-DTS surveys of temperature signals higher and lower than ambient temperature, ranging from well above to critically below the FO-DTS sampling interval. Our results reveal that qualitative and quantitative accuracies of FO-DTS measured temperatures critically decline with decreasing signal size, in particular for signals near the spatial sampling interval. Decreasing detection accuracy risks the masking of real temperature variation in highly dynamic systems. The resulting potential ambiguity of interpretations of signal size, intensity, and absolute location will have to be considered in future experimental design and interpretation of FO-DTS surveys.