Orthogonal time frequency space (OTFS) modulation is an emerging technology to tackle time-frequency (TF) selective channel in high mobility scenarios. In OTFS, resource is multiplexed in the delay-Doppler (DD) domain. Based on the potential sparsity, separability, stability and compactness of the channel spreading function, OTFS is able to realize lower complexity of channel estimation, higher diversity and higher reliability compared with orthogonal frequency division multiplexing (OFDM). However, the channel spreading function for practical communication systems is rarely considered in the current OTFS-related literature. High-speed railway (HSR) is a typical high mobility scenario with trains travelling at over 200km/h, which has the potential to employ OTFS. To this end, the HSR channel spreading function is characterized and the performance of OTFS in HSR is evaluated based on the realistic channel measurement in this article. Firstly, the HSR channel in TF domain is measured based on the long term evolution railway (LTE-R) network. Then, the characteristics of the channel spreading function are analyzed. In particular, the impact of time domain channel fading on the spreading function is investigated. The characteristics of the measured spreading function are analyzed with the proposed metrics in railway viaduct and tunnel scenarios. Based on the above analysis, an algorithm for generating the channel spreading function is proposed. Feasibility of the proposed DD domain channel generation algorithm is verified through comparing metrics of which to those of the measured channel. By simulating the bit error rate (BER) and mean square channel estimation error performances of OTFS modulation in the practical band-limited systems, it is shown that the impacts of Doppler shift, delay, SFFT and time domain channel fading need be considered for the application of OTFS modulation, in contrast to the state-of-art DD domain channel generation scheme based on tap delay link (TDL) model. For example, compared to OTFS modulation under the ideal channel spreading function, OTFS modulation requires a signal gain greater than 5 dB under the practical channel spreading function affected by above factors, to achieve the same BER less than 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-2</sup> under the parameters and algorithms given in section V.