Many subjective factors can affect energy cost (V’O2max, maximal strength and flexibility) and mechanical work (stride frequency) of walking. To our knowledge, no studies have been conducted to investigate if the level of daily physical activity (PAL) can affect external mechanical work (WEXT) and net energy cost (CW). The aim of the study was to analyse the relation between CW and WEXT with PAL. 20 healthy adults (age, 27,2±4 yr; BMI, 22,4±1,9 kg/m2; RMR 1758,3±261,3 kJ/d; V’O2max, 42±6,2 mlO2/kg/min) were recruited in the study and were classified as inactive (INACT) and active (ACT) according to the amount of daily moderate and vigorous physical activity (MVPA). Net CW was analysed with indirect calorimetry (K4b2, Cosmed, Italy) and simultaneously, a kinematic analysis was performed with an optoelectronic system (SMART-E, BTS, Italy) to calculate WEXT during 3 bouts of treadmill walking of 10 min each at 3,5/4,5/5,5 km/h. To assess PAL, subjects wore an activity monitor (Actiheart, CamNtech, UK) for a whole week, inferring time spent in sedentary (SED, <1,5 METs), or moderate to vigorous (MVPA, >3 METs) physical activity. When compared with INACT, ACT have a significantly higher amount of MVPA (P<0,0001). No group differences were observed for SED behaviour. ACT have higher values of both CW and WEXT than INACT (CW: 3,5 km/h ACT 2,56±0,31, INACT 2,5±0,37; 4,5 km/h ACT 2,57±0,38, INACT 2,53±0,42; 5,5 km/h ACT 2,93±0,54, INACT 2,87±0,47 J/kg/m; WEXT: 3,5 km/h ACT 0,3±0,08, INACT 0,26±0,04; 4,5 km/h ACT 0,27±0,07, INACT 0,24±0,05; 5,5 km/h ACT 0,25±0,07, INACT 0,22±0,05 J/kg/m). Significant correlations between CW or WEXT calculated at the different speeds were found(P<0,01). No significant associations were found between MVPA or SED with neither CW nor WEXT. Our data indicate that ACT and INACT adults differ for MVPA but not for SED patterns. None of these factors seem to influence CW and WEXT of treadmill walking of light intensity in a healthy adult population.