Respiration rate allows to differentiate between happy and sad excerpts which may be attributable to entrainment of respiration to the rhythm or the tempo rather than to emotions [Etzel, J.A., Johnsen, E.L., Dickerson, J., Tranel, D., Adolphs, R., 2006. Cardiovascular and respiratory responses during musical mood induction. Int. J. Psychophysiol. 61(1), 57-69]. In order to test for this hypothesis, this study intended to verify whether fast and slow rhythm, and/or tempo alone are sufficient to induce differential physiological effects. Psychophysiological responses (electrodermal responses, facial muscles activity, blood pressure, heart and respiration rate) were then measured in fifty young adults listening to fast/happy and slow/sad music, and to two control versions of these excerpts created by removing pitch variations (rhythmic version) and both pitch and temporal variations (beat-alone). The results indicate that happy and sad music are significantly differentiated (happy>sad) by diastolic blood pressure, electrodermal activity, and zygomatic activity, while the fast and slow rhythmic and tempo control versions did not elicit such differentiations. In contrast, respiration rate was faster with stimuli presented at fast tempi relative to slow stimuli in the beat-alone condition. It was thus demonstrated that the psychophysiological happy/sad distinction requires the tonal variations and cannot be explained solely by entrainment to tempo and rhythm. The tempo entrainment exists in the tempo alone condition but our results suggest this effect may disappear when embedded in music or with rhythm.