Seminal fluid has a role in conception and establishing pregnancy beyond simply delivery of sperm to fertilise the oocyte. In women, seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. This response is believed to be important in establishing female immune tolerance to paternal antigens associated with sperm and expressed by any ensuing embryo and fetus, thereby facilitating successful pregnancy. We have identified TGFB, an abundant cytokine in human seminal fluid, as a key mediator of this response. Previous studies have sought to examine whether relationships exist between TGFB, other seminal fluid cytokines and fertility status in men. Many of these studies have failed to identify any significant associations however this may be due in part to evaluation of only a single ejaculate from each subject. In this study, we assessed the within- and between-individual variability in seminal fluid cytokine concentrations over time, to determine the utility of measuring these factors in single semen samples. Semen samples were collected from 14 proven fertile donors approximately every 2 months, over a 12 month period. The concentrations of TGFB superfamily members TGFB1, TGFB2, TGFB3, Activin A and Follistatin were determined using specific ELISAs. Average variation from mean values within individuals was TGFB1 (14%), TGFB2 (25%), TGFB3 (27%), Activin A (33%) and Follistatin (42%). In addition, sperm concentration varied by 24%, motility by 5%, morphology by 33% and volume by 16%. Variation in each moiety was then assessed using a random effects model where it was observed that between-subject variation accounted for the majority of the variation observed, except for TGFB1 and Activin A, which did not display significant inter-individual variability. Our study suggests there is a need to assess multiple semen samples to assess soluble factors in human seminal fluid.