Importance: Previous research showed that a twice-daily 108 mg dose of calcium fructoborate (CFB) improved knee discomfort during a 2-week supplementation period. This current double-blind, placebo-controlled randomized study investigates the effects of CFB supplementation on knee discomfort during 90 days of supplementation. Purpose: To evaluate the comparative effects of once-daily and twice-daily dosing of calcium fructoborate on knee joint discomfort for ninety days. Design: 120 participants with self-reported knee discomfort were recruited and randomized into three groups (each N=40). Participants received: 108 mg CFB twice per day (CFB-G1); or, 216 mg CFB in a single dose (CFB-G2); or, placebo. Setting: Subjects were recruited through advertisement in local papers. The researchers assessed intake and within-study levels of knee discomfort by using the McGill Pain Questionnaire (MPQ) and the Western Ontario and McMaster University Arthritis Index (WOMAC). Results: 62 female and 59 male subjects completed the study. Subjects’ average age was (52.84 ± 8.19 years) and average BMI was (26.76 ± 2.50 kg/m2). Statistical differences between groups were calculated using a two-sided, two-sample t-test. Analysis of variance (ANOVA) was used to estimate within-group changes in mean WOMAC and MPQ scores as well as against the control group. When compared to placebo, CFB-G1 showed a significant decrease in reported discomfort on day 14 (P=0.02,) day 30 (P=0.003), day 60 (P<0.0001) and day 90 (P<0.0001) according to WOMAC Scores. A similar decrease was observed for CFB-G2 WOMAC Scores on day 14 (P=0.02), day 30 (P=0.0003), day 60 (P<0.0001) and day 90 (P<0.0001). When compared to placebo, the MPQ score for CFB-G1 group decreased on day 7 (P=0.002), day 14 (P=0.001), day 30 (P<0.0001), day 60 (P<0.0001) and day 90 (P<0.0001). MPQ score decreases were also observed for CFB-G2 group on day 7 (P=0.02), day 14 (P=0.01), day 30 (P<0.0001), day 60 (P<0.0001) and day 90 (P<0.0001). When comparing CFB-G1 and CFB-G2, no significant differences were observed. Importantly, no changes were observed in the WOMAC and MPQ scores within the placebo group. Conclusion: Both CFB groups showed early and significantly improved levels of knee comfort. Knee comfort continued to significantly improve throughout the duration of this 90-day study. No significant differences were observed between the once-daily and the twice–daily doses of CFB.