In this paper, 400 Gbps Tests - which take place in ITU's Roadmap of 2018 and have been never tried on a live network, according to literature review- are applied to Turk Telekom Live Network, within a distance of 1060/2120 kilometers (between Istanbul and Ankara, round-trip/between Istanbul - Ankara, twice round-trip) and changes on Optical Signal-to-Noise Ratio (OSNR) are observed by changing the number of channels on the transmission line, channel spacing, noise, modulation technique and output power. Additionally, two signals having frequencies of 10 and 40 GHz are coded by different modulation techniques (Non-Return Zero-NRZ for 10 GHz, Quadrature Phase-Shift Keying-QPSK for 40 GHz) and carried sequentially on the transmission line, with different channel spacing parameters, to investigate the effect of channel spacing to the transmission performance. The results show that, compared to 16-Quadrature Amplitude Modulation-QAM, QPSK performs better with an amount of nearly 90%, performance of choosing the channel spacing value as 50 GHz instead of 37.5 GHz is better, adding noise poisons the system's performance with a portion of 25% and increasing the output power leads nonlinear effect to grow. Finally, while carrying two signals having frequencies of 10 and 40 GHz with different channel spacing values, adjusting channel spacing as 100 GHz performs better with a portion of 6%, compared to 50 GHz.