The purpose of this study was to examine the role of active muscle mass on cardiovascular drift (CV_drift) during prolonged exercise. Twelve subjects with peak oxygen uptake (VO_2peak) of 3.52 ± 0.52 L·min^-1 (mean ± SD) cycled for 55 min with 80 revolutions per minute with either two legs (2-legged) or one leg (1-legged). Oxygen uptake was at 60% of VO_2peak throughout the 2-legged trial and at half of this value in 1- legged condition. Cardiac output (CO-CO₂ rebreathing), heart rate (HR) and quadriceps integrated electromyographic activity (iEMG) were higher (p < 0.01) during 2-legged than 1- legged exercise. Changes in stroke volume from 20 to 50 min of exercise were greater in 2-legged than in 1-legged (∆SV: -20.8 ± 0.8 vs. -13.3 ± 1.3 ml·beat^-1, p < 0.05). Similarly, changes in heart rate (∆HR) were +18.5 ± 0.8 and +10.7 ± 1.0 beats·min^-1, in 2-legged and 1-legged, respectively (p < 0.01). Calculated blood volume changes declined significantly in 2-legged exercise (∆BV: -4.25 ± 0.43%, p < 0.05). Sympathetic activation as indicated by the ratio of low and high frequency in spectral analysis of HR (LF HF^-1 ratio) was higher in 2-legged than in 1- legged trial (p < 0.05). At the end of exercise, CO had a tendency to decrease from 20^th min in 2-legged (changes in CO = -0.92 ± 0.3 L·min^-1, p = 0.07), whereas it was maintained in 1- legged cycling (∆CO = -0.15 ± 0.2 L·min^-1, p = 0.86). Multiple regression analysis showed that HR rise and blood volume decline were predictors of SV drop whereas heart rate increase was explained by rectal temperature and magnitude of muscle mass activation, as indicated by iEMG (p < 0.05) in 2-legged cycling. In conclusion, apart from the well-known factors of thermal status and blood volume decline, it seems that muscle mass involved plays also a role on the development of CV_drift.