PurposeLarge inter-individual variations in post-bariatric fat-free mass loss (FFML) are observed, which might relate to differences in protein intake and physical activity across patients. We performed repetitive assessments of protein intake and physical activity before and after banded Roux-en-Y gastric bypass surgery, and examined its relations to FFML during 6 months of follow-up.Materials and MethodsFFML (bio-impedance analyses), protein intake (24-h dietary recalls) and moderate-to-vigorous physical activity (MVPA; activPAL) were assessed in 28 patients (4 males, age 42 ± 12 years) before surgery and at 1-, 3- and 6-months post-surgery. Changes in protein intake and MVPA were evaluated with mixed model analysis, whereas associations with FFML were assessed by univariate regression analysis.ResultsSix-month FFML was -7.3 ± 3.6 kg. Protein intake decreased from 80 ± 29 g/day (pre-surgery) to 45 ± 26 g/day (1 month post-surgery (P< 0.001)) and did not improve thereafter (51 ± 21 g/day;P> 0.05). Seven participants (25%) consumed ≥ 60 g protein/day at 6 months post-surgery. Participants performed 7394 ± 2420 steps/day in 54 ± 20 min/day of MVPA, which did not change from pre- to post-surgery (P> 0.05). A higher step count (B = -0.002; 95%CI = [-0.004 – 0.000];P= 0.048) and higher level of MVPA (B = -0.29; 95%CI = [-0.54 – -0.03];P= 0.018) were related to a lower FFML.ConclusionA lower post-surgery FFML was attributable to higher MVPA levels but not protein intake. This may be due to the low total protein intake and the observation that only a minority of patients achieved a protein intake ≥ 60 g/day. Future studies should focus on interventions to increase post-bariatric protein intake and MVPA levels.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s11695-023-06650-y.