We examined the effects of gender and age in young and middle-aged subjects on the level of cardio-respiratory interaction by analyzing properties of cardiac, respiratory and cardiac-respiratory regulatory mechanisms under paced breathing. In 56 healthy subjects, ECG (RR interval) and respiratory signal were simultaneously acquired in supine position at paced (0.1-0.45 Hz, steps of 0.05 Hz) and spontaneous breathing. The participants were divided into gender matched group of young adults (19-25 years old) and middle-aged adults (35-44 years old). Power spectral analysis was applied on RR interval time series and spectral components in very low frequency (VLF), low frequency (LF) and high frequency (HF) ranges were computed. We also calculated sample entropy of RR interval series (SampEnRR), respiratory series (SampEnResp), and their cross-sample entropy (cross-SampEn). Under paced breathing, reduction of all spectral powers with age (p<0.05) is not gender dependent but reduction of some entropy measures is; SampEnRR and SampEnResp were lower only in men (p<0.05). In the middle-aged subjects, effect of gender on spectral measures is significant; males had lower HF (p < 0.05). Pattern of dependencies of SampEn and cross-SampEn on paced breathing frequency were significantly different in men (young vs. middle-aged, p = 0.001 and p = 0.037) and in middle-aged subjects (females vs. males, p = 0.011 and p = 0.008). In middle-aged males, lower entropy measures indicated reduced and less complex partial cardiac and respiratory control, and central cardio-respiratory control. In conclusion, in healthy middle-aged subjects changes in cardio-respiratory coupling are detectable only in males.