Exposure to a micro-gravity (μG) environment, particularly in the absence of counter measures, is known to induce a loss of skeletal muscle mass and function. We have recently used supine whole-body unloading on a hyper-saline filled water bed (hyper buoyancy floatation, HBF) as an analogue of microgravity (μG) and demonstrated an average ~1kg loss of total muscle mass after 7 days of HBF unloading1 as determined by whole-body magnetic resonance imaging (MRI). Here we have compared the loss of muscle determined by the gold standard (MRI) with that predicated by the creatine dilution (D3-creatine, D3-cr) as described by Clarke et al. (2014)2. Twelve healthy male subjects aged (27.3±4.2 yrs) completed the study. Six weeks prior to unloading each subject underwent a one-week control period. Pre and post the control period and at standardised time of day subjects undertook an MRI (Siemens MAGNETOM Verio 3T, Germany). For the unloading intervention period the subjects were asked to lie supine on the HBF for 7 days Subjects were allowed a maximum of 15 mins per day when they were not on the HBF (for personal hygiene etc) and were fed a controlled diet for both the control and intervention period. One day prior to and 1.5-3hrs post-unloading, further scans were performed. To estimate muscle mass using the D3-cr, after an overnight fast, subjects provided a baseline urine sample followed by a single 60 mg oral dose of D3-cr (two 30 mg capsules) at ~08:00 h on day 3 of the control and day 3 of the unloading period. Total urine was collected from baseline, through to the same recorded dosage time (~08:00 h) on Day 5. Measurements of urine creatine, creatinine, D3-cr and D3-creatinine, were performed by liquid chromatography/mass spectrometry. No significant changes were observed in MRI-derived muscle mass before and after the control period, and D3-cr muscle mass was similar to mean value of the two MRI measures (31.8±5.1 v 33.3±12.7 kg (mean+ SD); p=0.309). The unloading period induced a ~1kg loss of muscle mass MRI (32.19±5.33 versus 31.25±5.33 kg; p=0.0002). However, D3-cr predicted an 8.5kg decrease in muscle mass between the control and unloading period (31.8±5.1 v 23.3+7.4 kg; p=0.0001), which was significantly different to the post-unloading muscle measured using MRI (23.3+7.4 kg v 31.25±5.33 kg; p=0.0081). The values for D3-cr and pre and post unloading MRI were correlated (r2 = 0.303; p=0039 and r2 = 0.295 ; p=0.0081, respectively), but the change in muscle mass determined by MRI was not correlated with change determined by D3-cr (r2 = 0.01, ns) The results showed that whilst the D3-cr method correlated with MRI predictions of total skeletal muscle mass, compared to the gold standard measure (MRI) the D3-cr method markedly overestimated muscle loss induced by 7 days of unloading.