A new physiological assessment method was presented by novel use of photoplethysmography (PPG) signals. Most previous PPG signal processing techniques were based on time and frequency domain characteristics or wave form of this signal In the present study, time differences between separate and simultaneous PPG signal pairs with different wavelengths were compared as the novel aspect of PPG technology. A computer connectable multi-channel PPG prototype device and a MATLAB (R) based program were developed to obtain four concurrent transmission mode PPG signals with wavelengths of 940nm (infrared), 660nm (red), 520nm (green) and 450nm (blue). The time differences between different pairs of these signals were obtained during a breath holding experiment to investigate and compare their variations within the experiments. Results showed that time differences of some PPG signal pairs had significant variations in reply to the breath holding processes (as physiological challenges). It was concluded that PPG pair with wavelengths of 940nm and 660nm had the greatest reaction to the physiological changes.